Anti-bacterial applications of poly-N-acetylglucosamine nanofibers

ABSTRACT

Described herein are compositions comprising shortened fibers of poly-N-acetylglucosamine and/or a derivative thereof (“sNAG nanofibers”) and anti-bacterial applications of such compositions. The sNAG nanofibers may be formulated into compositions for the prevention and/or treatment of bacterial infections and diseases associated with such infections. Regimens employing such compositions are also described.

This application is a divisional of U.S. application Ser. No.13/641,015, filed Oct. 12, 2012, which is a U.S. national stage entry ofInternational Application No. PCT/US2011/032709, filed Apr. 15, 2011,which claims the benefit of U.S. Provisional Application No. 61/324,657,filed Apr. 15, 2010, each of which is incorporated herein by referencein its entirety.

1. FIELD

Described herein are compositions comprising shortened fibers ofpoly-N-acetylglucosamine and/or a derivative thereof (“sNAG nanofibers”)and anti-bacterial applications of such compositions. The sNAGnanofibers may be formulated into compositions for the prevention and/ortreatment of bacterial infections and diseases associated with suchinfections. Regimens employing such compositions are also described.

2. BACKGROUND

Currently, antibiotics are a standard therapy for bacterial infections.However, some individuals have an allergic reaction to certainantibiotics, others suffer from side effects associated withantibiotics, and the continued use of antibiotics often leads to areduction in their efficacy. In addition, antibiotic therapy often leadsto the emergence of antibiotic-resistant strains of bacteria.Accordingly, there is a continuing need for new anti-bacterial agentsthat are effective in fighting infection without generating resistanceor reducing the efficacy overtime. There is a need for non-antibioticanti-bacterial agents that can be used in clinical settings, e.g., inthe treatment of infectious diseases of the skin, digestive andrespiratory tract, and in wound treatment.

Wound infection is one type of bacterial infection. Wound infection is amajor complication, especially in patients with chronic disease such asdiabetes or during immunosuppression. Such patients have disruptions inappropriate inflammatory responses, including the migration andrecruitment of neutrophils and macrophages, which predisposes them toincreased infection (Singer, A. J. and R. A. Clark, 1999, N Engl J Med341(10): 738-46). In addition, bacterial infection can lead toimpairment of wound healing and sepsis. Given the ineffectiveness ofmany current antibiotic treatments and the increased prevalence ofantibiotic resistant bacteria such as MRSA (Methycillin-resistant S.aureus), new clinical treatments are in high demand.

3. SUMMARY

In one aspect, described herein are methods for treating and/orpreventing a bacterial infection(s) and/or diseases associated with orcaused by a bacterial infection in a subject.

In certain embodiments, described herein are methods for treating abacterial infection in a subject comprising topically administering acomposition comprising sNAG nanofibers to a subject. In someembodiments, the subject is diagnosed with the bacterial infection ordisplaying one or more symptoms of the bacterial infection. The methodsof diagnosis of bacterial infection and symptoms of bacterial infectionare those known in the art or described herein. The bacterial infectionmay be a skin infection, a gastrointestinal infection, a respiratoryinfection, a urinary tract infection, a reproductive tract infection, orinfection of any other organ or tissue in the body of the subject asdescribed herein. In one embodiment, the infection is a nosocomialinfection, an MRSA infection, a Pseudomonas infection, or a C. dificuleinfection.

In certain embodiments, described herein are methods for treating and/orpreventing a disease associated with a bacterial infection or abacterial imbalance in a subject comprising topically administering acomposition comprising sNAG nanofibers to the subject. In one suchembodiment, the method involves treating and/or preventing a diseaseassociated with a bacterial infection. In another embodiment, the methodinvolves treating and/or preventing a disease associated with abacterial imbalance, for example, an imbalance in bacterial microbiotaas described herein. In certain embodiments, the methods involvetreating an existing bacterial infection. In some of these embodiments,the subject to be treated is diagnosed with a disease associated with abacterial infection or displays one or more symptoms of such disease. Inother embodiments, the subject to be treated is diagnosed with a diseaseassociated with a bacterial imbalance or displays one or more symptomsof such imbalance. The disease may be a skin disease, a gastrointestinaldisease, a respiratory disease, a urinary tract disease, a reproductivetract disease, or disease of any other organ or tissue in the body ofthe subject as described herein. In some embodiments, the disease is askin disease or a gastrointestinal disease. In one embodiment, thedisease is associated with a nosocomial infection, an MRSA infection, aPseudomonas infection, or a C. dificule infection.

In some embodiments, described herein are methods for preventing abacterial infection and/or a disease associated with a bacterialinfection comprising topically administering a composition comprisingsNAG nanofibers to a subject. In some embodiments, a compositioncomprising sNAG nanofibers is administered to a subject at high risk ofa bacterial infection to prevent a disease associated with a bacterialinfection. In specific embodiments, a composition comprising sNAGnanofibers is administered to a subject with a wound or a subject whohas undergone a surgery. In one embodiment, the composition isadministered to an immunocompromised subject. In some embodiments, acomposition comprising sNAG nanofibers is administered to a wound, wherethe wound is at high risk of bacterial infection. In certainembodiments, the wound is an open wound. The open wound may be a gunshotwound, a puncture wound, a laceration wound, an abrasion, a cut, apenetration wound, a surgical wound, or any other wound. In certainembodiments, the wound may be a puncture wound, for example, a puncturewound that is caused by a hemodialysis procedure or a catheterizationprocedure. In such embodiments, the subject to be treated may have beendiagnosed with a hemodialysis-related or catheterization-relatedinfection. In one embodiment, the bacterial infection and/or the diseaseassociated with a bacterial infection to be prevented by a sNAGcomposition is not in a wound (e.g., an open wound) or is not associatedwith a wound. In one such embodiment, the bacterial infection and/or thedisease associated with a bacterial infection is not at the site of awound (e.g., not at the site of an open wound).

In some embodiments, described herein are methods for treating abacterially infected wound in a subject, comprising topicallyadministering a composition comprising sNAG nanofibers to the wound sitein a subject. In some embodiments, the subject to be treated isdiagnosed with a bacterial infection or displays one or more symptoms ofthe bacterial infection. In certain embodiments, the wound is an openwound. The open wound may be a gunshot wound, a puncture wound, alaceration wound, an abrasion, a cut, a penetration wound, a surgicalwound, or any other wound. In certain embodiments, the wound may be apuncture wound, for example, a puncture wound that is caused by ahemodialysis procedure or a catheterization procedure. In suchembodiments, the subject to be treated may have been diagnosed with ahemodialysis-related or catheterization-related infection.

Bacterial infections to be treated or prevented using the methodsdescribed herein include infections with bacteria of one or more of thefollowing genuses: Bordetella, Borrelia, Brucella, Campylobacter,Chlamnydia and Clamidophylia, Clostridium, Corynebacterium,Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter,Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus,Streptococcus, Treponema, Vibria, and Yersinia. In some embodiments, asNAG composition may be used to treat and/or prevent a diseaseassociated with an infection by bacteria from one or more of the listedgenuses of bacteria, or one or more symptoms thereof.

Bacterial infections to be treated or prevented using the methodsdescribed herein also include infections with bacteria of one or more ofthe following species: Bacillus anthracis, Bordetella pertussis,Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucellamelitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia,Chlamydia trachomatis, Clamidophila psittaci, Clostridium botulinum,Clostridium dificule, Clostridium perfringens, Clostridium tetani,Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Francisella tularensis, Haemophilusinfluenae, Helicobacter pylori, Legionella pneumphila, Leptospirapneumophila, Leptospira interrogans, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa,Proteus mirabilis, Rickettsia rickettsii, Salmonella typhi, Salmonellatyphimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus; Streptococcus agalactiae,Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum,Vibria cholerae, and Yersinia pestis. In some embodiments, a sNAGcomposition may be used to treat and/or prevent a disease associatedwith an infection by bacteria from one or more of the listed species ofbacteria, or one or more symptoms thereof.

In certain embodiments, the bacterial infection to be treated orprevented using the methods described herein is an MRSA infection, aPseudomonas infection, or a C. dificule infection. In some embodiments,a sNAG composition may be used to treat and/or prevent a diseaseassociated MRSA infection, a Pseudomonas infection, or a C. dificuleinfection, or one or more symptoms thereof symptom thereof.

In certain embodiments, the bacterial infection to be treated orprevented using the methods described herein is caused by bacteria thatare known to one of ordinary skill in the art to be resistant to astandard anti-bacterial therapy, for example, resistant to one or moreantibiotics. In one embodiment, the bacterial infection to be treated orprevented using the methods described herein is MRSA, e.g., a nosocomialMRSA. In some embodiments, a sNAG composition may be used to treatand/or prevent a disease associated with an infection by bacteriaresistant to one or more antibiotics. In one embodiment, a sNAGcomposition may be used to treat and/or prevent a disease associatedwith MRSA, e.g., associated with a nosocomial MRSA.

The subject to be treated using the methods described herein may be amammal, preferably a human. The subject can also be a livestock animal(e.g., a chicken, a cow, a pig, a goat) or a pet (e.g., a dog or a cat),or any other animal.

The sNAG nanofibers contemplated in the methods described herein may beof varying lengths, widths and molecular weights as described in Section5.1, infra. In certain embodiments, the majority (and in certainembodiments, at least or more than 60%, 70%, 80%, 90%, 95% or 99%) ofthe sNAG nanofibers, or 100% of the sNAG nanofibers, are between about 1to 15 μm in length. In some embodiments, the majority (and in certainembodiments, at least or more than 60%, 70%, 80%, 90%, 95% or 99%) ofthe sNAG nanofibers, or 100% of the sNAG nanofibers, are between about 2to 10 μm, or 4 to 7 μm in length. The sNAG nanofibers of the describedlength can be obtained, for example, as described below in Section 5.2,infra.

In certain embodiments, the sNAG nanofibers were produced byirradiation, e.g., gamma irradiation, of poly-N-acetylglucosamine or aderivative thereof. In some embodiments, the sNAG nanofibers areproduced by irradiation of the poly-β-1→4-N-acetylglucosamine in theform of dried fibers (e.g., at 500-2,000 kgy), or irradiation of thepoly-β-1→4-N-acetylglucosamine in the form of wet fibers (e.g., at100-500 kgy).

In certain embodiments, the sNAG nanofibers are derived from microalgae.In another embodiment, the sNAG nanofibers are not derived fromcrustaceans. In yet another embodiment, the sNAG nanofibers may bederived from microalgae, crustaceans (e.g., shrimp), fungus or any othersource.

In one embodiment, the sNAG nanofibers comprise N-acetylglucosaminemonosaccharides and/or glucosamine monosaccharides, wherein more than60%, 70%, 80%, 90%, 95%, or 99% of the monosaccharides of the sNAGnanofibers are N-acetylglucosamine monosaccharides. In anotherembodiment, the sNAG nanofibers comprise N-acetylglucosaminemonosaccharides and/or glucosamine monosaccharides, wherein more than70% of the monosaccharides of the sNAG nanofibers areN-acetylglucosamine monosaccharides.

In certain embodiments, the sNAG nanofibers used in the methodsdescribed herein do not have an effect on bacterial growth or survivalof Staphylococcus aureus bacterial cultures in vitro, or substantiallyhave no effect on bacterial growth or survival of Staphylococcus aureusbacterial cultures in vitro. In some embodiments, the sNAG nanofibersreduce bacterial growth or survival of bacterial cultures in vitro byless than 1 log, 0.75 log, 0.5 log, 0.25 log, 0.2 log or 0.1 log, e.g.,when Staphylococcus aureus bacterial cultures are treated/incubated withthe sNAG nanofibers in vitro. The tests for the effect of sNAGnanofibers on bacterial growth or survival and the evaluation of thetest results are described, for example, in Section 5.1, Example 2(e.g., Section 6.2.2.5) and FIG. 11E, infra.

In certain embodiments, the sNAG nanofibers used in the methodsdescribed herein are non-reactive in a biocompatibility test or tests.For example, the sNAG nanofibers used in the methods described hereinmay be non-reactive when tested in an elution test, an intramuscularimplantation test, an intracutaneous test, or a systemic test. In someembodiments, the compositions described herein are non-reactive whentested in an elution test, an intramuscular implantation test, anintracutaneous test, or a systemic test. In other embodiments, the sNAGnanofibers used in the methods described herein have Grade 0 or Grade 1when tested in an elution test, an intramuscular implantation test, anintracutaneous test, or a systemic test. In yet another embodiment, thesNAG nanofibers used in the methods described herein are at most mildlyreactive when tested in an elution test, an intramuscular implantationtest, an intracutaneous test, or a systemic test. In one embodiment, thesNAG nanofibers or compositions comprising such nanofibers arenon-reactive as determined by an intramuscular implantation test. Incertain embodiments, the compositions described herein do not cause anallergenic reaction or an irritation, e.g., at the site of application.In other embodiments, the compositions described herein cause at most amild allergenic reaction or a mild irritation, e.g., at the site ofapplication.

The contemplated modes of administration of the compositions describedherein are topical, e.g., topical on the skin; topical at the site of awound, a surgery, a bacterial infection, or a symptom of an infection(e.g., a swelling); and topical to a body surface such as the skin,mucous membranes (e.g., vagina, anus, throat, eyes, ears), or thesurface of other tissues. In certain embodiments, the sNAG nanofibers orcompositions comprising such nanofibers are formulated as a dressing, abandage, a mat, a spray, a liquid, a suspension, a membrane, a powder,an ointment, a cream, a paste, a suppository, or a gel. In someembodiments, the sNAG nanofibers or compositions comprising suchnanofibers are formulated as a cream, a gel, an ointment, a membrane, apowder, a spray, or a suppository.

In another aspect, described herein are compositions for use in themethods described herein. In a specific embodiment, the compositionscomprise sNAG nanofibers. In certain embodiments, the compositionsdescribed herein comprise sNAG nanofibers and one or more additionalactive ingredients useful in preventing and/or treating a bacterialinfection, a disease associated with a bacterial infection, or a symptomthereof. In some embodiments, the additional active ingredient is ananti-bacterial agent. Such additional anti-bacterial agent may be anantibiotic. In another embodiment, such additional anti-bacterial agentis zinc. In yet another embodiment, the compositions described herein donot comprise an antibiotic. In yet other embodiments, the compositionsdescribed herein do not comprise any additional anti-bacterial agent. Inone embodiment, the compositions described herein comprise the sNAGnanofibers as the only active ingredient and do not comprise anyadditional active ingredients.

In specific embodiments, a composition comprises the sNAG nanofibers andan antibiotic. Examples of antibiotics that can be used in thecompositions of the invention include microlides (e.g., erythromycin,azithromycin), aminoglycosides (e.g., amikacin, gentamicin, neomycin,streptomycin), cephalosporins (e.g., cefadroxil, cefaclor, cefotaxime,cefepime), fluoroquinolones (e.g., ciprofloxacin, levofloxacin),penicillins (e.g., penicillin, ampicillin, amoxicillin), tetracyclines(e.g., tetracycline, doxycycline), and/or carbapenems (e.g., meropenem,imipenem). The sNAG nanofibers and agents described herein may be usedin such compositions. In some embodiments, a composition comprises thesNAG nanofibers and an agent effective to treat or prevent or commonlyused to treat or prevent an S. aures infection, MRSA infection, aPseudomonas infection, or a C. dificule infection (e.g., an antibioticeffective against or commonly used against such infections).

In other embodiments, the compositions described herein are administeredin conjunction with one or more additional anti-bacterial agents or anyother suitable therapy. In some embodiments, the additionalanti-bacterial agent or therapy is an antibiotic (e.g., a standardantibiotic therapy for the bacterial infection or a disease associatedwith a bacterial infection to be treated, as known in the art ordescribed herein). In some embodiments, the additional anti-bacterialagent is an agent effective to treat or prevent or commonly used totreat or prevent an S. aures infection, MRSA infection, a Pseudomonasinfection, or a C. dificule infection (e.g., an antibiotic effectiveagainst or commonly used against such infections). In some embodiments,the additional therapy is administered before, simultaneously with orafter administration of a sNAG nanofiber composition. In yet anotherembodiment, the compositions described herein are not administered inconjunction with any other therapy, e.g., not administered inconjunction with an antibiotic.

3.1 TERMINOLOGY

As used herein, the terms “sNAG nanofiber,” “sNAG,” “Taliderm,” or“Talymed” (formerly known as “Taliderm”) are used interchangeably torefer to shortened fibers of poly-N-acetylglucosamine and/or derivativesthereof.

As used herein, the term “about” means a range around a given valuewherein the resulting value is the same or substantially the same (e.g.,within 10%, 5% or 1%) as the expressly recited value. In one embodiment,“about” means within 10% of a given value or range. In anotherembodiment, the term “about” means within 5% of a given value or range.In another embodiment, the term “about” means within 1% of a given valueor range.

As used herein, the terms “disease,” “disorder” or “condition” are usedinterchangeably to refer to a medical condition in a subject. In aspecific embodiment, the disease is the pathological state associatedwith or caused by a bacterial infection.

As used herein, the term “bacterial infection” means the invasion by,multiplication and/or presence of bacteria in a cell or a subject.

As used herein, the numeric term “log” refers to log₁₀.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compositions, formulations, and/or agent(s) thatcan be used in the prevention and/or treatment of a bacterial infectionor a symptom or condition associated therewith. In certain embodiments,the term “therapy” refers to a sNAG nanofiber(s) or a pharmaceuticalcomposition comprising a sNAG nanofiber(s). In other embodiments, theterm “therapy” refers to a therapy other than a sNAG nanofiber(s) or apharmaceutical composition comprising a sNAG nanofiber(s). In specificembodiments, an “additional therapy” and “additional therapies” refer toa therapy other than a sNAG nanofiber(s) or a pharmaceutical compositioncomprising a sNAG nanofiber(s). In a specific embodiment, the therapyincludes use of a sNAG nanofiber(s) or pharmaceutical compositioncomprising a sNAG nanofiber(s) as an adjuvant therapy; for example,using a sNAG nanofiber composition in conjunction with a drug therapy,such as an antibiotic, and/or other therapies useful in treatment and/orprevention of a bacterial infection or a symptom or condition associatedtherewith.

As used herein, the term “effective amount” in the context ofadministering a sNAG nanofiber composition to a subject refers to theamount of a sNAG nanofiber that results in a beneficial or therapeuticeffect. In specific embodiments, an “effective amount” of a sNAGnanofiber refers to an amount which is sufficient to achieve at leastone, two, three, four or more of the following effects: (i) theclearance of a bacterial infection; (ii) the eradication of one or moresymptoms associated therewith, (iii) the reduction of time required toclear a bacterial infection; (iv) the reduction or amelioration of theseverity of a bacterial infection and/or one or more symptoms associatedtherewith; (v) the reduction in the duration of a bacterial infectionand/or one or more symptoms associated therewith; (vi) the prevention ordelay of the generation of a resistant strain or strains of bacteria orreduction of a number of resistant strains of bacteria generated; (vii)the prevention in the recurrence of a bacterial infection and/or one ormore symptoms associated therewith; (viii) the reduction or eliminationin the bacterial cell population; (ix) the reduction in the severityand/or duration of a condition caused by or associated with a bacterialinfection; (x) the reduction in hospitalization of a subject; (xi) thereduction in hospitalization length; (xii) the increase in the survivalof a subject; (xiii) the enhancement or improvement of the therapeuticeffect of another therapy; (xiv) a reduction in mortality; (xv) thereduction or elimination in the spread of the bacteria from one subjectto another subject, or one organ or tissue to another organ or tissue;(xvi) the prevention of an increase in the number of bacteria; (xvii)the prevention of the development or onset of a bacterial infection orone or more symptoms associated therewith; (xviii) the reduction in thenumber of symptoms associated with a bacterial infection; (xix) thereduction in the duration and/or severity of a condition caused by orassociated with a bacterial infection; (xx) the inhibition or reductionin production of a bacterial toxin or toxins associated with a bacterialinfection; (xxi) the stabilization or reduction of inflammationassociated with a bacterial infection: (xxii) the induction of theexpression of one or more defensin proteins and/or defensin-likeproteins; (xxiii) the induction of the expression of one or moreToll-like receptors; (xxiv) the induction of the expression of one ormore proteins that are beneficial for clearance or reduction in abacterial infection or one or more symptoms associated therewith; (xxvi)the reduction in organ failure associated with a bacterial infection ora disease associated therewith; (xxvii) the prevention of the onset,development or recurrence of a condition caused by or associated with abacterial infection; and/or (xxviii) improvement in quality of life asassessed by methods well known in the art, e.g., a questionnaire. Inspecific embodiments, an “effective amount” of a sNAG nanofiber refersto an amount of a sNAG nanofiber composition specified herein, e.g., inSection 5.6, infra.

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human infant” refers to a newborn to 1 yearold year human.

As used herein, the term “premature human infant” refers to a newborn to1 year old year human who was born of less than 37 weeks gestational age(e.g., before 37 weeks, 36 weeks, 35 weeks, 34 weeks, 33 weeks, 32weeks, 31 weeks, 30 weeks, 29 weeks, 28 weeks, or less than 28 weeks ofpregnancy).

As used herein, the term “human toddler” refers to a human that is 1year to 3 years old.

As used herein, the term “majority” refers to greater than 50%,including, e.g., 50.5%, 51%, 55%, etc.

As used herein, the term “subject” and “patient” are usedinterchangeably to refer to an animal (e.g., cow, horse, sheep, pig,chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc.).In a specific embodiment, the subject is a mammal such as a non-primateor a primate, e.g., a human. In specific embodiments, the subject is ahuman. See Section 5.5, infra, for more information concerning patientstreated in accordance with the methods provided herein.

As used herein, the term “low expression,” in the context of expressionof a gene (e.g., based on the level of protein or peptide produced bythe gene) refers to an expression that is less than the “normal”expression of the gene. In a specific embodiment, “low expression”refers to expression of a gene that is less than 90%, less than 80%,less than 75%, less than 70%, less than 65%, less than 60%, less than55%, less than 50%, less than 45%, less than 40%, less than 35%, lessthan 30%, less than 25%, or less than 20% of the “normal” expression ofthe gene. In another specific embodiment, “low expression” refers toexpression of a gene that is about 20-fold, about 15-fold, about10-fold, about 5-fold, about 4-fold, about 3-fold, about 2-fold, orabout 1.5 fold less than the “normal” expression of the gene.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. Nanofibers stimulate Akt1 activation, an upstream regulator ofEts1. (A) Western blot analysis of phospho-Akt in response to NAG andsNAG stimulation of serum starved EC. (B) RT-PCR analysis of EC infectedeither with scrambled control (“SCR”) or Akt1 shRNA lentiviruses andassessed for expression of Ets1 and S26 as a loading control. (C)Schematic of a signal transduction pathway transducing a signal fromsNAG nanofibers to Akt1, Ets1 and Defensins.

FIG. 2. Delayed wound healing in Akt1 null animals is partially rescuedby Taliderm treatment. (A) Representative images of wounded WT and AKT1null mice with and without treatment of Taliderm. (B) H&E staining ofrepresentative mouse skin sections from day 3 wounds.

FIG. 3. sNAG nanofibers stimulate cytokine and defensin expression inprimary endothelial cells. (A) Immunohistochemisty of EC treated with orwithout sNAG using an antibody directed against a-defensin. (B) ELISAshowing that nanofiber treatment of EC results in the secretion ofα-defensins 1-3 (serum starved, treated with 5 μg/ml or 10 μg/ml sNAG).

FIG. 4. sNAG nanofibers stimulate defensin expression in primaryendothelial cells in an Akt1 dependent manner. (A) and (B) QuantitativeRT-PCR analyses of serum starved EC (“ss”) treated with or without sNAG(“snag”), with or without PD98059 (MAPK inhibitor, “PD”), Wortmannin(PI3K inhibitor, “wtm”) or infected with a scrambled control (“SCR”), orAkt1 (“AKT1”) shRNA lentiviruses and assessed for expression of thegenes indicated.

FIG. 5. sNAG nanofibers stimulate β-defensin 3 expression in mousekeratinocytes. (A) Immunofluorescent staining with β-defensin 3 (visibleas bright staining in the upper right hand panel; see, e.g., thick whitearrows) and Involucrin antibodies of paraffin embedded mouse cutaneouswound sections from WT and Akt1 null animals on Day 3. (B)Quantification of β-defensin 3 immunofluorescent staining usingNIHImageJ software (TX=Taliderm; Akt1=Akt1 null). (C) Immunofluorescentstaining of WT and Akt1 null treated and untreated keratinocytes withβ-Defensin 3 (visible as bright staining; see, e.g., thick white arrows)and TOPRO-3 (nuclei staining; see, e.g., thin white arrows). Notice theincrease in β-Defensin 3 staining in WT and Akt1 Taliderm treatedwounds.

FIG. 6. Akt1 dependent transcription factor binding sites. Schematic ofAkt1 dependent transcription factor binding sites. Using Genomatixsoftware, 500 bp upstream of the transcription start site was analyzedfor conserved sites on the mRNA of DEF1, 4, and 5 (ETS-black ovals;FKHD-striped ovals; CREB-white ovals; NFKB-checkered ovals).

FIG. 7. sNAG treatment results in expression and secretion of defensinsin vitro. (A) RTPCR analysis of serum starved (“SS”) primary endothelialcells treated with sNAG (50 μg/ml) for the times indicated and assessedfor expression of β-defensin 3 and α-defensin 1. (B) Immunofluorescentlabeling of endothelial cells either serum starved (untreated) ortreated with sNAG nanofibers (10 μg/ml for 5 hrs). Antibodies aredirected against α-defensin 5 (FITC, upper left hand panel), β-defensin3 (Texas Red, upper right hand panel). Nuclei are stained with TOPRO-3(Blue, lower left hand panel). Lower right hand panel represents tripleoverlay. (C) Immunofluorescent labeling of keratinocytes (HaCat) thatare either serum starved (untreated) or treated with sNAG nanofibers (10μg/ml for 5 hours). Antibodies are directed against α-defensin 5 (FITC,upper left hand panel), β-defensin 3 (Texas Red, upper right handpanel). Nuclei are stained with TOPRO-3 (Blue, lower left hand panel).

FIG. 8. sNAG induced defensin expression is dependent on Akt1. (A)Quantitative RT-PCR analyses using primers directed against α-defensin 1from total RNA isolated from serum starved endothelial cells treatedwith or without sNAG for 3 hours, with or without pretreatment withPD098059 (“PD”)(50 μM), wortmannin (“WTM”)(100 nm). Quantitation isrelative to the S26 protein subunit. (B) Quantitation of β-defensin 3expression from total RNA isolated from serum starved endothelial cellstreated with or without sNAG for 3 hours, with or without PD98059 (50μm), wortmannin (100 nm) and shown as relative to S26. (C) Western Blotanalysis of phospho-Akt in serum starved endothelial cells (SS)stimulated with sNAG for the times indicated. Line indicates where laneshave been removed (D) Quantitative RT-PCR analyses of serum starvedendothelial cells infected with a scrambled control (SCR) or Akt1 shRNAlentiviruses, treated with or without sNAG and assessed for α-defensin 4expression. Quantitation is shown relative to S26. (E) Quantitation ofβ-defensin 3 expression from total RNA isolated from serum starvedendothelial cells infected with a scrambled control (SCR) or Akt1 shRNAlentiviruses, treated with or without sNAG. Quantitation is shownrelative to 526. All experiments were done in at least triplicate andrepeated at least three independent times and p values are shown.

FIG. 9. sNAG induced defensin expression in vivo requires Akt1. (A)Paraffin embedded sections of cutaneous wounds harvested on day 3 postwounding from both WT (n=3) and Akt1 mice. Wounds were either untreatedor treated with sNAG membrane. Immunofluorescence was performed usingantibodies directed against β-defensin 3 (green, visible as brightstaining in the upper right hand panel; see, e.g., white thick arrows),Involucrin (Red), and Topro (Blue, nuclei staining; see, e.g., whitethin arrows). (B) Paraffin embedded section from WT treated with sNAGharvested on day 3. Immunofluorescence was performed using antibodiesdirected against β-defensin 3 (green, visible as bright staining; see,e.g., thick white arrows), Involucrin (Red), and Topro (Blue, nucleistaining; see, e.g., thin white arrows). This lower magnification (20×)is included to better illustrate the epidermal layers expressingf-defensin 3. Scale bars=50 μm. (C) Quantitation of β-defensin 3expression from paraffin embedded sections was performed using NIHImageJ software. Experiments were repeated three independent times and pvalues are shown.

FIG. 10. sNAG treatment increases wound closure in wild type mice. H&Estaining of wound tissue sections derived from C57Bl6 wild type animalseither untreated or treated with sNAG membrane. The day post-wound isindicated to the left of each panel. The solid black line follows thekeratinocyte cell layer indicating wound closure. Black arrows indicatethe margin of the wound bed.

FIG. 11. sNAG treatment reduces bacterial infection in an Akt1 dependentmanner. (A) Tissue gram staining of S. aureus infected wounds from WTmice. WT mice were wounded using a 4 mm biopsy punch. Immediately afterwounding mice were inoculated with 1×10⁹ cfu/ml. 30 minutespost-infection, mice in the treated group were treated with Taliderm.Skin samples were taken 5 days post-treatment and sectioned foranalysis. Tissue gram staining was performed. Dark purple stainingindicates gram-positive bacteria and neutrophils that have engulfedbacteria. Sections under 20× and 40× magnification are shown. (B) Tissuegram staining of paraffin embedded S. aureus infected wounds from WT andAkt1 null mice (n=3). Infected wounds were either untreated or treatedwith sNAG membrane and wound beds were harvested on day 3 and day 5 foranalysis. Dark purple staining indicates the presence of gram positivebacteria in the wound bed. Black arrows indicate examples of grampositive staining. Note the accumulation of positive staining inuntreated WT that is lacking in WT animals treated with sNAG. Scalebars=50 μm. (C) CFUs derived from day 5 post wounding were quantitatedfrom S. aureus infected wounds using both treated and untreated WT (n=3)and Akt1 mice (n=3). Wild type mice that were sNAG treated show asignificant (p<0.01) decrease in bacteria load in the wound beds ascompared to Akt1 null animals. All experiments were repeated threeindependent times and the p values are shown. (D) CFU quantitated frominfected wounds at day 3 post wounding in a similar fashion described in(C). sNAG treatment of infected wounds shows a significant decrease inCFU of both WT and Akt1 null animals on day 3, but the WT animals showan approximate 10 fold difference compared to a 2 fold difference inAkt1 animals. (E) Quantitation of CFUs in S. aureus cultures that wereeither untreated or treated with various amounts of sNAG nanofibers.Each experiment was performed three independent times and p values areshown. (F) Tissue gram staining of S. aureus infected wounds harvestedon day 3 post wound from WT mice (n=3) that were treated with or withoutβ-defensin 3 peptide (1.0 uM). Note the decrease in gram positivestaining in infected wounds that were treated with β-defensin 3 peptide.(G) Quantitation of CFUs from S. aureus infected WT mice (n=3) treatedwith or without β-defensin 3 peptide. Infected wounds that were treatedwith peptide show a significant decrease (p<0.05) in CFU. Scale bars=50μm. Each experiment was performed three independent times and p valuesare shown.

FIG. 12. Rapid induction of defensin expression by sNAG treatment of S.aureus infected wounds. (A) Paraffin embedded tissue sections from S.aureus infected wounds, harvested on day 3, were subjected toimmunofluorescence using antibodies directed against β-defensin 3(green, visible as bright staining in the upper right hand panel and inthe lower panel in the middle; see, e.g., thick white arrows),Involucrin (red) to mark the keratinocyte layer, and Topro (blue, nucleistaining; see, e.g., thin white arrows) from both sNAG treated WT (n=3)and untreated WT mice (n=3). Non specific staining of keratin isindicated by the no primary control which was stained with secondaryantibody only. Scale bar=50 μm. (B) Quantitation of β-defensin 3expression from paraffin embedded sections using NIH ImageJ software. S.aureus infected wounds that were treated with sNAG show a significantincrease (p<0.05) in β-defensin 3 staining. Experiments were repeatedthree independent times and p values are shown.

FIG. 13. Antibodies against β-defensin 3 impedes antibacterial effectsof sNAG treatment. (A) Tissue gram staining of paraffin embedded S.aureus infected wounds treated with sNAG from WT mice (n=3) that wereharvested on Day 3. sNAG treated wounds were treated with eitherβ-defensin 3 antibody or isotype control goat IgG antibody prior to sNAGtreatment. Representative images show increased accumulation grampositive staining (black arrows) in the wound beds of mice treated withan antibody directed against β-defensin 3. Scale bar=20 μm. (B)Quantitation of CFUs from S. aureus infected WT mice treated eitherβ-defensin 3 antibody (n=3) or control IgG antibody (n=3) prior to sNAGtreatment. β-defensin 3 application significantly increased (p<0.05)CFU.

FIG. 14. sNAG treatment reduces bacterial infection by Pseudomonasaeruginosa. Mice were wounded using a 4 mm biopsy punch, inoculated with1.5×10⁹ cfu/ml P. aeruginosa, infected wounds were either untreated(n=6) or treated (n=6) with sNAG membrane (n=6) 30 min post-infection,wound beds were harvested on day 3 for analysis, cultured for 30minutes, plated, and CFUs of the untreated and treated infected woundswere quantitated. sNAG treated mice show a significant (p<0.05) decreasein bacteria load in the wound beds as compared to untreated animals.

FIG. 15. Effect of irradiation on chemical and physical structure ofpGlcNAc fibers. (A) Correlation between molecular weight of pGlcNAc andirradiation level/formulation for irradiation. (B) Infrared (IR)spectrum of non-irradiated pGlcNAc slurry (top line), pGlcNAc slurryirradiated at 100 kGy (bottom line), and pGlcNAc slurry irradiated at200 kGy (middle line). (C) Scanning electron microscopic (SEM) analysesof pGlcNAc. (D) Scanning electron microscopic (SEM) analyses of sNAG.

FIG. 16. pGlcNAc did not affect metabolic rate. For each time period(i.e., at 24 and 48 hours), the identity for each of the four bars (fromleft to right) is as follows: serum starvation (SS), VEGF, and pGlcNAc(NAG) at 50 and 100 μg/ml.

FIG. 17. pGlcNAc protected human umbilical vein endothelial cell (EC)from cell death induced by serum deprivation. For each time period(i.e., at 24, 48 and 72 hours), the identity for each of the five bars(from left to right) is as follows: serum starvation (SS), VEGF, andpGlcNAc (NAG) at 50, 100, and 250 μg/ml.

FIG. 18. sNAG induced marked increase in metabolic rate. Identity foreach of the five bars (from left to right) is as follows: serumstarvation (SS), VEGF, and sNAG at 50, 100 and 200 μg/ml.

FIG. 19. sNAG did not protect EC from cell death induced by serumdeprivation. For each time period (i.e., at 24 and 48 hours), theidentity for each of the five bars (from left to right) is as follows:serum starvation (SS), VEGF, and sNAG at 50, 100 and 200 μg/ml.

5. DETAILED DESCRIPTION

The inventors have discovered that sNAG nanofibers decrease bacterialinfection of cutaneous wounds infected with Staphylococcus aureus andPseudomonas aeurginosa. Without being bound by any specific mechanism ofaction, data presented in Section 6.2 suggests that the antibacterialeffect of sNAG is not due to a direct interaction of sNAG with thebacteria but is due to downstream affects such as, for example, theregulation of defensins by Akt1 activation. Specifically, data show thattreatment of bacterial cultures with sNAG nanofibers in vitro does notaffect bacterial count indicating that sNAG nanofibers do not directlyinhibit bacterial growth. In a specific example described in Section6.2.2.5 and illustrated in FIG. 11E, sNAG nanofibers do not have adirect effect on growth or survival of Staphylococcus aureus. The testdescribed in Section 6.2.2.5, infra, may be used to test the lack of adirect effect of sNAG nanofibers on bacterial growth or survival. Inthis example, S. aureus cultures in solution were treated with varyingconcentrations of sNAG nanofibers for three hours, cultures were thenplated overnight at 37° C. and bacterial CFU/ml determined. As shown inFIG. 11E, no effect on bacterial growth or survival was observed.

The inventors of the present invention have found that sNAG nanofiberscan stimulate expression of defensins, which may boost the innateanti-bacterial response. It is widely accepted that defensins areimportant players in innate immunity and function in anti-bacterialactivities. As demonstrated in the examples presented in Sections 6.1and 6.2, infra, the inventors of the present invention have found thatsNAG nanofibers can increase the expression of both α- and β-typedefensins in endothelial cells and β-type defensins in keratinocytes invitro and in a wound healing model in vivo.

Further, as demonstrated in the examples presented in Sections 6.1 and6.2, infra, but without being bound by any specific mechanism of action,Akt1 appears to be important for sNAG-dependent defensin expression invitro and in vivo, in a wound healing model. Consistently, sNAGtreatment decreased bacterial infection of cutaneous wounds infectedwith Staphylococcus aureus in wild type control animals but not insimilarly treated Akt1 null animals.

The inventors of this invention have also found that a number ofToll-like receptors can be up-regulated by sNAG treatment of humanendothelial cells. Toll-like receptors (“TLRs” or “TLR”) are highlyconserved receptors that recognize specific molecular patterns ofbacterial components leading to activation of innate immunity. Recentwork has linked human defensin expression to TLR activation. Inparticular, stimulation of TLRs can lead to increased defensinsynthesis. Thus, without being bound by any mechanism of action, sNAGnanofibers may act as a stimulator of innate immunity and bacterialclearance via the activation of Akt1.

Accordingly, described herein is the use of sNAG nanofibers as a novelmethod for preventing and/or treating bacterial infections and diseasesassociated therewith. In certain embodiments, treatment of bacterialinfections with sNAG nanofibers decreases the bacterial load inpatients. In specific embodiments, the use of sNAG nanofibers enhanceswound closure while simultaneously eradicating, decreasing or preventingbacterial infection of the wound.

5.1 sNAG Nanofibers

Described herein are sNAG nanofiber compositions. The sNAG nanofiberscomprise fibers of poly-N-acetylglucosamine and/or a derivative(s)thereof, the majority of which are less than 30 microns in length and atleast 1 micron in length as measured by any method known to one skilledin the art, for example, by scanning electron microscopy (“SEM”). SuchsNAG nanofibers may be obtained, for example, as described herein.

In certain embodiments, the majority (and in certain embodiments, atleast 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%,or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%,80% to 95%, 90% to 95%, or 95% to 99%) of the sNAG nanofibers are lessthan about 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 microns inlength, and at least 1 micron in length as measured by any method knownto one skilled in the art, for example, by SEM. In specific embodiments,the majority (and in certain embodiments, at least 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between 55% to 65%, 55%to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or95% to 99%) of the sNAG nanofibers are less than about 15 microns orless than about 12 microns in length, and at least 1 micron in length asmeasured by any method known to one skilled in the art, for example, bySEM. In specific embodiments, all (100%) of the sNAG nanofibers are lessthan about 15 microns in length, and at least 1 micron in length asmeasured by any method known to one skilled in the art, for example, bySEM. In certain embodiments, the majority (and in certain embodiments,at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or100%, or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the sNAG nanofibers areequal to or less than 14, 13, 12, 11, 10, 9, 8 or 7 microns in length,and at least 1 micron in length as measured by any method known to oneskilled in the art, for example, by SEM. In some embodiments, themajority (and in certain embodiments, at least 60%, 70%, 80%, 90%, 95%,98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between 55% to 65%, 55% to75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95%to 99%) of the sNAG nanofibers are between 1 to 15, 2 to 15, 2 to 14, 1to 12, 2 to 12, 1 to 10, 2 to 10, 3 to 12, 3 to 10, 1 to 9, 2 to 9, 3 to9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 1 to6, 1 to 5, 1 to 4, or 1 to 3 microns in length as measured by any methodknown to one skilled in the art, for example, by SEM.

In a specific embodiment, the majority (and in certain embodiments, atleast 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%,or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%,80% to 95%, 90% to 95%, or 95% to 99%) of the sNAG nanofibers are about8, 7, 6, 5, 4, 3 or 2 microns in length as measured by any method knownto one skilled in the art, for example, by SEM. In another specificembodiment, the majority (and in certain embodiments, at least 60%, 70%,80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between 55% to65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to95%, or 95% to 99%) of the sNAG nanofibers are between about 2 to about10 microns, about 3 to about 8 microns, or about 4 to about 7 microns inlength as measured by any method known to one skilled in the art, forexample, by SEM. In another specific embodiment, all (100%) of the sNAGnanofibers are between about 2 to about 10 microns, about 3 to about 8microns, or about 4 to about 7 microns in length as measured by anymethod known to one skilled in the art, for example, by SEM.

In certain embodiments, the sNAG nanofibers fibers are in a rangebetween 0.005 to 5 microns in thickness and/or diameter as determined byelectron microscopy. In specific embodiments, the sNAG nanofibers areabout 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6,2.8, 3 or 4 microns in thickness and/or diameter on average, or anyrange in between (e.g., 0.02 to 2 microns, 0.02 to 1 microns, 0.02 to0.75 microns, 0.02 to 0.5 microns, 0.02 to 0.5 microns, 0.05 to 1microns, 0.05 to 0.75 microns, 0.05 to 0.5 microns, 0.1 to 1 microns,0.1 to 0.75 microns, 0.1 to 0.5 microns, etc.). In specific embodiments,the majority (and in certain embodiments, at least 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between 55% to 65%, 55%to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or95% to 99%) of the sNAG nanofibers have a thickness or diameter of about0.02 to 1 microns. In other specific embodiments, the majority (and incertain embodiments, at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%,99.8%, 99.9%, or 100%, or between 55% to 65%, 55% to 75%, 65% to 75%,75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95% to 99%) of thesNAG nanofibers have a thickness or diameter of about 0.05 to 0.5microns. In specific embodiments, all (100%) of the sNAG nanofibers havea thickness or diameter of about 0.02 to 1 microns or about 0.05 to 0.5microns. In certain embodiments, the majority (and in certainembodiments, at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%,99.9%, or 100%, or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the sNAGnanofibers have a thickness or diameter of about 0.02 to 2 microns, 0.02to 1 microns, 0.02 to 0.75 microns, 0.02 to 0.5 microns, 0.02 to 0.5microns, 0.05 to 1 microns, 0.05 to 0.75 microns, 0.05 to 0.5 microns,0.1 to 1 microns, 0.1 to 0.75 microns, or 0.1 to 0.5 microns.

In certain embodiments, the majority (and in certain embodiments, atleast 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%,or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%,80% to 95%, 90% to 95%, or 95% to 99%) of the sNAG nanofibers arebetween 1 and 15 microns in length and have a thickness or diameter ofabout 0.02 to 1 microns.

In certain embodiments, the molecular weight of the sNAG nanofibers isless than 100 kDa, 90 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55kDa, 50 kDa, 45 kDA, 40 kDa, 35 kDa, 30 kDa, or 25 kDa. In certainembodiments, the majority (and in certain embodiments, at least 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%,90% to 95%, or 95% to 99%) of the sNAG nanofibers have a molecularweight of less than 100 kDa, 90 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60kDa, 55 kDa, 50 kDa, 45 kDA, 40 kDa, 35 kDa, 30 kDa, or 25 kDa. In otherembodiments, the majority (and in certain embodiments, at least 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%,90% to 95%, or 95% to 99%) of the sNAG nanofibers have a molecularweight between about 5 kDa to 100 kDa, about 10 kDa to 100 kDa, about 20kDa to 100 kDa, about 10 kDa to 80 kDa, about 20 kDa to 80 kDa, 20 kDato 75 kDa, about 25 kDa to about 75 kDa, about 30 kDa to about 80 kDa,about 30 kDa to about 75 kDa, about 40 kda to about 80 kDa, about 40 kDato about 75 kDa, about 40 kDa to about 70 kDa, about 50 kDa to about 70kDa, or about 55 kDa to about 65 kDa. In one embodiment, the majority(and in certain embodiments, at least 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.5%, 99.8%, 99.9%, or 100%, or between 55% to 65%, 55% to 75%, 65% to75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95% to 99%) ofthe sNAG nanofibers have a molecular weight of about 60 kDa.

In certain embodiments, 1% to 5%, 5% to 10%, 5% to 15%, 20% to 30% or25% to 30% of the sNAG nanofibers are deacetylated. In some embodiments,1%, 5%, 10%, 15%, 20%, 25%, or 30% of the sNAG nanofibers aredeacetylated. In other embodiments, less than 30%, 25%, 20%, 15%, 10%,5%, 4%, 3%, 2% or 1% of the sNAG nanofibers are deacetylated. In someembodiments, equal to or more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, orall (100%), of the sNAG nanofibers are deacetylated. In otherembodiments, less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the sNAGnanofibers are deacetylated.

In certain embodiments, 70% to 80%, 75% to 80%, 75% to 85%, 85% to 95%,90% to 95%, 90% to 99% or 95% to 100% of the sNAG nanofibers areacetylated. In some embodiments, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%or 100% of the sNAG nanofibers are acetylated. In other embodiments,more than 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of thesNAG nanofibers are acetylated. In some embodiments, equal to or morethan 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%, or all (100%), of the sNAGnanofibers are acetylated. In other embodiments, less than 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, or 100% of the sNAG nanofibers are acetylated.

In some embodiments, the sNAG nanofibers comprise at least oneglucosamine monosaccharide, and may further comprise at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the N-acetylglucosaminemonosaccharides. In other embodiments, the sNAG nanofibers comprise atleast one N-acetylglucosamine monosaccharide, and may further compriseat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% ofglucosamine monosaccharides.

In one aspect, the sNAG nanofibers increase the metabolic rate ofserum-starved human umbilical cord vein endothelial cells (“EC”) in aMTT assay. A MTT assay is a laboratory test and a standard colorimetricassay (an assay which measures changes in color) for measuring cellularproliferation (cell growth). Briefly, yellow MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, atetrazole) is reduced to purple formazan in the mitochondria of livingcells. This reduction takes place only when mitochondrial reductaseenzymes are active, and therefore conversion can be directly related tothe number of viable (living) cells. The metabolic rate of cells may bedetermined by other techniques commonly known to the skilled artisan.

In another aspect, the sNAG nanofibers do not rescue apoptosis ofserum-starved EC in a trypan blue exclusion test. A trypan blueexclusion test is a dye exclusion test used to determine the number ofviable cells present in a cell suspension. It is based on the principlethat live cells possess intact cell membranes that exclude certain dyes,such as trypan blue, Eosin, or propidium, whereas dead cells do not. Theviability of cells may be determined by other techniques commonly knownto the skilled artisan.

In certain embodiments, compositions comprising the sNAG nanofibers aredescribed, wherein the sNAG nanofibers increase the metabolic rate ofserum-starved human umbilical cord vein endothelial cells in a MTT assayand/or do not rescue apoptosis of serum-starved human umbilical cordvein endothelial cells in a trypan blue exclusion test. In someembodiments, the sNAG nanofibers increase the metabolic rate ofserum-starved human umbilical cord vein endothelial cells in a MTT assayand do not rescue apoptosis of serum-starved human umbilical cord veinendothelial cells in a trypan blue exclusion test.

In a specific embodiment, the sNAG nanofibers are biocompatible.Biocompatibility may be determined by a variety of techniques,including, but not limited to such procedures as the elution test,intramuscular implantation, or intracutaneous or systemic injection intoanimal subjects. Such tests are described in U.S. Pat. No. 6,686,342(see, e.g., Example 10), which is incorporated by reference herein inits entirety.

In certain embodiments, the sNAG nanofibers used in the methodsdescribed herein are non-reactive in a biocompatibility test or tests.For example, the sNAG nanofibers used in the methods described hereinmay be non-reactive when tested in an elution test, an intramuscularimplantation test, an intracutaneous test, and/or a systemic test. Inother embodiments, the sNAG nanofibers used in the methods describedherein have Grade 0 or Grade 1 test score when tested in an elutiontest, an intramuscular implantation test, an intracutaneous test, or asystemic test. In yet another embodiment, the sNAG nanofibers used inthe methods described herein are at most mildly reactive when tested inan elution test, an intramuscular implantation test, an intracutaneoustest, and/or a systemic test. In certain embodiments, the compositionsdescribed herein do not cause an allergenic reaction or an irritation.In other embodiments, the compositions described herein cause at most amild allergenic reaction or a mild irritation, e.g., at the site ofapplication. The relevant tests and evaluation of test results aredescribed in, e.g., U.S. Pat. No. 6,686,342, which is incorporatedherein by reference in its entirety, and in Section 6.8, infra.

In a specific embodiment, the sNAG nanofibers are non-reactive whentested in an intramuscular implantation test. In one aspect, anintramuscular implantation test is an intramuscular implantationtest—ISO 4 week implantation, as described in Section 6.8.3, infra. Incertain embodiments, the sNAG nanofibers display no biologicalreactivity as determined by an elution test (Elution Test Grade=0). Insome embodiments, the sNAG nanofibers have a test score equal to “0”and/or are at most a negligible irritant as determined by intracutaneousinjection test. In some embodiments, the sNAG nanofibers elicit nointradermal reaction (i.e., Grade I reaction) in Kligman test and/orhave a weak allergenic potential as determined by Kligman test.

In certain aspects, the sNAG nanofibers are immunoneutral (i.e., they donot elicit an immune response).

In some embodiments, the sNAG nanofibers are biodegradable. The sNAGnanofibers preferably degrade within about 1 day, 2 days, 3 days, 5days, 7 days (1 week), 8 days, 10 days, 12 days, 14 days (2 weeks), 17days, 21 days (3 weeks), 25 days, 28 days (4 weeks), 30 days, 1 month,35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 2 months, 65 days,70 days, 75 days, 80 days, 85 days, 90 days, 3 months, 95 days, 100 daysor 4 months after administration or implantation into a patient.

In certain embodiments, the sNAG nanofibers do not cause a detectableforeign body reaction. A foreign body reaction, which may occur duringwound healing, includes accumulation of exudate at the site of injury,infiltration of inflammatory cells to debride the area, and theformation of granulation tissue. The persistent presence of a foreignbody can inhibit full healing. Rather than the resorption andreconstruction that occurs in wound healing, the foreign body reactionis characterized by the formation of foreign body giant cells,encapsulation of the foreign object, and chronic inflammation.Encapsulation refers to the firm, generally avascular collagen shelldeposited around a foreign body, effectively isolating it from the hosttissues. In one embodiment, treatment of a site (e.g., a wound or a siteof a bacterial infection in a wound) with the sNAG nanofibers does notelicit a detectable foreign body reaction in 1 day, 3 days, 5 days, 7days, 10 days or 14 days after treatment. In one such embodiment,treatment of a site (e.g., a wound) with the sNAG nanofibers does notelicit a foreign body encapsulations in 1 day, 3 days, 5 days, 7 days,10 days or 14 days after treatment.

In some embodiments, the sNAG nanofibers (i) comprise fibers, whereinmajority of the fibers are between about 1 and 15 microns in length, and(ii) (a) increase the metabolic rate of serum-starved EC in a MTT assayand/or do not rescue apoptosis of serum-starved EC in a trypan blueexclusion test, and (b) are non-reactive when tested in an intramuscularimplantation test. In certain embodiments, the sNAG nanofibers (i)comprise fibers, wherein majority of the fibers are between about 1 and12 microns in length, and (ii) (a) increase the metabolic rate ofserum-starved EC in a MTT assay and/or do not rescue apoptosis ofserum-starved EC in a trypan blue exclusion test, and (b) arenon-reactive when tested in an intramuscular implantation test. Incertain embodiments, the sNAG nanofibers (i) comprise fibers, whereinmajority of the fibers are between about 4 and 7 microns in length, and(ii) (a) increase the metabolic rate of serum-starved EC in a MTT assayand/or do not rescue apoptosis of serum-starved EC in a trypan blueexclusion test, and (b) are non-reactive when tested in an intramuscularimplantation test.

In certain embodiments, the sNAG nanofibers do not have a direct effecton the growth or survival of bacteria, such as S. aureus, as determinedby one skilled in the art. In other embodiments, sNAG nanofibers do nothave a direct effect on the growth or survival of bacteria, such as S.aureus, as determined by the methods set forth in Section 6.2.2.5,infra. In some embodiments, the sNAG nanofibers do not have a directeffect in vitro on bacterial growth or survival. In one embodiment, thesNAG nanofibers do not have a direct effect (e.g., in vitro) on growthor survival of gram-negative bacteria. In another embodiment, the sNAGnanofibers do not have a direct effect (e.g., in vitro) on growth orsurvival of gram-positive bacteria. In yet another embodiment, the sNAGnanofibers do not have a direct effect (e.g., in vitro) on growth orsurvival of either gram-positive or gram-negative bacteria. In someembodiments, the sNAG nanofiber or a sNAG nanofiber composition does notbind bacteria (e.g., gram-positive bacteria, gram-negative bacteria, orboth types of bacteria). In some embodiments, incubation of a bacterialculture with the sNAG nanofibers (e.g., 50-500 μg of sNAG nanofibers) invitro does not reduce bacterial load in 10 minutes, 30 minutes, 1 hour,2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours or 96hours of incubation (wherein the bacterial culture may be gram-positiveand/or a gram-negative). In some embodiments, incubation of a S. aureusculture with sNAG nanofibers (e.g., about 80 μg-300 μg, or about 100-200μg of sNAG nanofibers) in vitro does not reduce bacterial load in 2hours, 3 hours, 6 hours or 24 hours of incubation. In yet otherembodiments, the sNAG nanofibers reduce bacterial growth or survival invitro by less than 1 log, 0.9 log, 0.8 log, 0.75 log, 0.7 log, 0.6 log,0.5 log, 0.4 log, 0.3 log, 0.25 log, 0.2 log, 0.1 log, 0.05 log, or0.025 log, for example, when Staphylococcus aureus bacterial culturesare treated/incubated with the sNAG nanofibers in vitro. In someembodiments, the sNAG nanofibers reduce bacterial growth or survival invitro by less than 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴,8×10⁴, 9×10⁴, or 10×10⁴ cfu/ml, for example, when Staphylococcus aureusbacterial cultures are treated/incubated with the sNAG nanofibers invitro. The tests of the effect of sNAG nanofibers on bacterial growth orsurvival and the evaluation of the test results are described, forexample, in Example 2 (e.g., Section 6.2.2.5) and FIG. 11E, infra.

In some embodiments, the sNAG nanofibers (i) comprise fibers, whereinmajority of the fibers are between about 1 and 15 microns, 1 and 12microns, or 4 and 7 microns in length, (ii) do not have an effect onbacterial growth or survival of Staphylococcus aureus bacterial culturesin vitro, and (iii) are non-reactive when tested in a biocompatibilitytest (e.g., an intramuscular implantation test).

In certain embodiments, the sNAG nanofibers induce a certain pattern ofgene expression (RNA or protein expression as determined by, e.g.,RT-PCR, microarray or ELISA) in a cell, tissue or organ treated with orexposed to a sNAG nanofiber composition. Specifically, in someembodiments, the sNAG nanofibers or a composition comprising the sNAGnanofibers induce expression of one or more defensin proteins, one ormore defensin-like proteins, and/or one or more Toll-like receptors. Inyet other embodiments, the sNAG nanofibers or a composition comprisingthe sNAG nanofibers induce expression of one or more proteins that areknown to have an anti-bacterial effect.

In certain embodiments, the sNAG nanofibers or a composition comprisingthe sNAG nanofibers induce expression of one or more α-defensins (e.g.,DEFA1 (i.e., α-defensin 1), DEFA1B, DEFA3, DEFA4, DEFA5, DEFA6), one ormore β-defensins (e.g., DEFB1 (i.e., β-defensin 1), DEFB2, DEFB4,DEFB103A, DEFB104A, DEFB105B, DEFB107B, DEFB108B, DEFB110, DEFB112,DEFB114, DEFB118, DEFB119, DEFB123, DEFB124, DEFB125, DEFB126, DEFB127,DEFB128, DEFB129, DEFB131, DEFB136), and/or one or more θ-defensins(e.g., DEFT1P). In some embodiments, the sNAG nanofibers or acomposition comprising the sNAG nanofibers induce expression of one ormore of DEFA1, DEFA3, DEFA4, DEFA5, DEFB1, DEFB3, DEFB103A, DEFB104A,DEFB108B, DEFB112, DEFB114, DEFB118, DEFB119, DEFB123, DEFB124, DEFB125,DEFB126, DEFB128, DEFB129 and DEFB131. In certain embodiments, the sNAGnanofibers or a composition comprising the sNAG nanofibers induceexpression of one or more Toll receptors (e.g., TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, and/or TLR12). In otherembodiments, the sNAG nanofibers or a composition comprising the sNAGnanofibers induce expression of one or more of IL-1, CEACAM3, SPAG11,SIGIRR (IL1-like receptor), IRAK1, IRAK2, IRAK4, TBK1, TRAF6 and IKKi.In some embodiments, the sNAG nanofibers or a composition comprising thesNAG nanofibers induce expression of one or more of IRAK2, SIGIRR, TLR1,TLR2, TLR4, TLR7, TLR8, TLR10 and TRAF6. In one embodiment, the sNAGnanofibers or a composition comprising the sNAG nanofibers induceexpression of at least one of the above-listed gene products.

In some embodiments, the sNAG nanofibers or a composition comprising thesNAG nanofibers induce expression of one or more of the above-listedgenes in the amount equal to or more than about 0.25 fold, 0.5 fold, 1fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 12 fold, 15 fold or 20fold as compared to the level of expression of the one or more of theabove-listed genes in a cell, tissue or organ of a subject beforetreatment with the sNAG nanofibers (e.g., a known average level ofexpression of the one or more of the above-listed genes). In someembodiments, the sNAG nanofibers or a composition comprising the sNAGnanofibers induce expression of one or more of the above-listed genes inthe amount equal to or more than about 10%, 25%, 50%, 75%, 100%, 125%,150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 450%, 500%, 550%,600%, 650%, 700%, 750%, 800%, 900% or 1000% the level of expression ofthe one or more of the above-listed genes in a cell, tissue or organ ofa subject before treatment with the sNAG nanofibers (e.g., a knownaverage level of expression of the one or more of the above-listedgenes).

In some embodiments, the sNAG nanofibers but not longpoly-N-acetylglucosamine, chitin and/or chitosan induce expression ofthe one or more genes listed above, as determined by a method known toone skilled in the art, or described herein. In some of theseembodiments, long poly-N-acetylglucosamine, chitin and/or chitosan donot induce expression of the one or more genes listed above or inducelower level (e.g., more than 1.25 fold, 1.5 fold, 2 fold, 2.5 fold, 3fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, or 10 fold lower) of expression of the one or more genes listedabove as compared to the level of expression of the one or more geneslisted above induced by the sNAG nanofibers, as determined by a methodknown to one skilled in the art, or described herein.

In certain embodiments, the sNAG nanofibers or a composition comprisingthe sNAG nanofibers induce a gene expression profile that is consistentwith, similar to, about the same as, or equivalent to one or more geneexpression profiles demonstrated in Tables I, II, III, V, VIII and IX,Sections 6.2-6.5, infra. In some embodiments, the sNAG nanofibers or acomposition comprising the sNAG nanofibers induce expression of one ormore of the genes shown to be upregulated by sNAG treatment in Tables I,II, III, V, VIII and IX, Sections 6.2-6.5, infra. In some embodiments,the sNAG nanofibers or a composition comprising the sNAG nanofibersinduce expression of the majority or all of the genes shown to beupregulated by sNAG treatment in Tables I, II, III, V, VIII and IX,Sections 6.2-6.5, infra. In some of these embodiments, gene expressionlevels are measured at 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 8hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24hours, 48 hours, 3 days or 5 days after treatment of a cell, tissue ororgan with a sNAG nanofiber composition by a method known to one skilledin the art, or described herein.

In certain embodiments, the sNAG nanofibers or a composition comprisingthe sNAG nanofibers induce a gene expression profile that differs fromthe profile induced by long poly-N-acetylglucosamine polymers or fibers.In specific embodiments, a gene expression profile induced by the sNAGnanofibers is consistent with, similar to, about the same as, orequivalent to that shown in Tables I, II, III, V, VIII and IX, Sections6.2-6.5, infra, whereas gene expression profile induced by longpoly-N-acetylglucosamine polymers or fibers is consistent with, similarto, about the same with, or equivalent to that shown in Table VII and/orIX, Section 6.5, infra. In other embodiments, the sNAG nanofibers or acomposition comprising the sNAG nanofibers induce a gene expressionprofile that differs from the gene expression profile induced by chitinor chitosan.

In a specific embodiment, the sNAG nanofibers are obtained byirradiating poly-N-acetylglucosamine and/or a derivative thereof. SeeSection 5.1.1, infra, regarding poly-N-acetylglucosamine and derivativesthereof and Section 5.2, infra, regarding methods for producing the sNAGnanofibers using irradiation. Irradiation may be used to reduce thelength of poly-N-acetylglucosamine fibers and/orpoly-N-acetylglucosamine derivative fibers to form shortenedpoly-β-1→4-N-acetylglucosamine fibers and/or shortenedpoly-N-acetylglucosamine derivative fibers, i.e. sNAG nanofibers.Specifically, irradiation may be used to reduce the length and molecularweight of poly-N-acetylglucosamine or a derivative thereof withoutdisturbing its microstructure. The infrared spectrum (IR) of sNAGnanofibers is similar to, about the same as, or equivalent to that ofthe non-irradiated poly-β-1→4-N-acetylglucosamine or a derivativethereof.

In one embodiment, the sNAG nanofibers are not derived from chitin orchitosan. Whereas in another embodiment, the compositions describedherein may be derived from chitin or chitosan, or the sNAG nanofibersmay be derived from chitin or chitosan.

5.1.1 Poly-N-Acetylglucosamine and Derivatives Thereof

U.S. Pat. Nos. 5,622,834; 5,623,064; 5,624,679; 5,686,115; 5,858,350;6,599,720; 6,686,342; 7,115,588 and U.S. Patent Pub. 2009/0117175 (eachof which is incorporated herein by reference) describe thepoly-N-acetylglucosamine and derivatives thereof, and methods ofproducing the same. In some embodiments, the poly-N-acetylglucosaminehas a β-1→4 configuration. In other embodiments, thepoly-N-acetylglucosamine has a α-1→4 configuration. Thepoly-N-acetylglucosamine and derivatives thereof may be in the form of apolymer or in the form of a fiber.

Poly-N-acetylglucosamine can, for example, be produced by, and may bepurified from, microalgae, preferably diatoms. The diatoms which may beused as starting sources for the production of thepoly-N-acetylglucosamine include, but are not limited to members of theCoscinodiscus genus, the Cyclotella genus, and the Thalassiosira genus.Poly-N-acetylglucosamine may be obtained from diatom cultures via anumber of different methods, including the mechanical force method andchemical/biological method known in the art (see, e.g., U.S. Pat. Nos.5,622,834; 5,623,064; 5,624,679; 5,686,115; 5,858,350; 6,599,720;6,686,342; and 7,115,588, each of which is incorporated herein byreference in its entirety). In certain embodiments, thepoly-N-acetylglucosamine is not derived from one or more of thefollowing: a shell fish, a crustacean, an insect, a fungi or yeasts.

In one embodiment, poly-β-1→4-N-acetylglucosamine is derived from aprocess comprising a) treating a microalgae comprising a cell body and apoly-β-1→4-N-acetylglucosamine polymer fiber with a biological agent(such as hydrofluoric) capable of separating the N-acetylglucosaminepolymer fiber from the cell body for a sufficient time so that thepoly-β-1→4-N-acetylglucosamine polymer fiber is released from the cellbody; b) segregating the poly-β-1→4-N-acetylglucosamine polymer fiberfrom the cell body; and c) removing contaminants from the segregatedpoly-β-1→4-N-acetylglucosamine polymer fiber, so that thepoly-β-1→4-N-acetylglucosamine polymer is isolated and purified.

In other embodiments, the poly-β-1→4-N-acetylglucosamine may be derivedfrom one or more of the following: a shell fish, a crustacean, aninsect, a fungi or yeasts. In certain embodiments, the compositionsdescribed herein do not comprise chitin or chitosan.

One or more of the monosaccharide units of the poly-N-acetylglucosaminemay be deacetylated. In certain embodiments, 1% to 5%, 5% to 10%, 5% to15%, 20% to 30% or 25% to 30% of the poly-N-acetylglucosamine isdeacetylated. In some embodiments, 1%, 5%, 10%, 15%, 20%, 25%, or 30% ofthe poly-N-acetylglucosamine is deacetylated. In other embodiments, lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of thepoly-N-acetylglucosamine is deacetylated. In some embodiments, equal toor more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or all (100%), of thepoly-N-acetylglucosamine is deacetylated. In other embodiments, lessthan 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of thepoly-N-acetylglucosamine is deacetylated.

In certain embodiments, a poly-N-acetylglucosamine composition comprises70% to 80%, 75% to 80%, 75% to 85%, 85% to 95%, 90% to 95%, 90% to 99%or 95% to 100% of acetylated glucosamine (i.e., N-acetylglucosamine)monosaccharides. In some embodiments, a poly-N-acetylglucosaminecomposition comprises 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% ofacetylated glucosamine (i.e., N-acetylglucosamine) monosaccharides. Inother embodiments, a poly-N-acetylglucosamine composition comprises morethan 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of theacetylated glucosamine. In some embodiments, a poly-N-acetylglucosaminecomposition comprises equal to or more than 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99%, or all (100%), of the acetylated glucosamine. In other embodiments,a poly-N-acetylglucosamine composition comprises less than 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, or 100% of the acetylated glucosamine.

In some embodiments, a poly-N-acetylglucosamine composition comprises atleast one glucosamine monosaccharide, and may further comprise at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% ofN-acetylglucosamine monosaccharides. In other embodiments, apoly-N-acetylglucosamine composition comprises at least oneN-acetylglucosamine monosaccharide, and may further comprise at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of glucosaminemonosaccharides.

Derivatives of poly-N-acetylglucosamine may also be used in acomposition described herein. Derivatives of poly-N-acetylglucosamineand methods of making such derivatives are described in U.S. Pat. No.5,623,064 (see, e.g., Section 5.4), which is incorporated by referenceherein in its entirety. Derivatives of poly-N-acetylglucosamine mayinclude, but are not limited to, partially or completely deacetylatedpoly-N-acetylglucosamine, or its deacetylated derivatives. Further,poly-N-acetylglucosamine may be derivatized by being sulfated,phosphorylated and/or nitrated. Poly-N-acetylglucosamine derivativesinclude, e.g., sulfated poly-N-acetylglucosamine derivatives,phosphorylated poly-N-acetylglucosamine derivatives, or nitratedpoly-N-acetylglucosamine derivatives. Additionally, one or more of themonosaccharide units of the poly-N-acetylglucosamine may contain one ormore sulfonyl groups one or more O-acyl groups. In addition, one or moreof the monosaccharides of the deacetylated poly-N-acetylglucosamine maycontain an N-acyl group. One or more of the monosaccharides of thepoly-N-acetylglucosamine or of its deacetylated derivative, may containan O-alkyl group. One or more of the monosaccharide units of thepoly-N-acetylglucosamine may be an alkali derivative. One or more of themonosaccharide units of the deacetylated derivative ofpoly-N-acetylglucosamine may contain an N-alkyl group. One or more ofthe monosaccharide units of the deacetylated derivative ofpoly-N-acetylglucosamine may contain at least one deoxyhalogenderivative. One or more of the monosaccharide units of the deacetylatedderivative of poly-N-acetylglucosamine may form a salt. One or more ofthe monosaccharide units of the deacetylated derivative ofpoly-N-acetylglucosamine may form a metal chelate. In a specificembodiment, the metal is zinc. One or more of the monosaccharide unitsof the deacetylated derivative of poly-N-acetylglucosamine may containan N-alkylidene or an N-arylidene group. In one embodiment, thederivative is an acetate derivative. In another embodiment, thederivative is not an acetate derivative. In one embodiment thepoly-N-acetylglucosamine or deacetylated poly-N-acetylglucosamine isderivatized with lactic acid. Wherein, in another embodiment, thederivative is not derivatized with lactic acid.

5.2 Methods of Making sNAG Nanofibers

The poly-N-acetylglucosamine polymers or fibers, and any derivatives ofpoly-N-acetylglucosamine polymers or fibers described above, can beirradiated as dry polymers or fibers or polymer or fiber membranes.Alternatively, poly-N-acetylglucosamine polymers or fibers, and anyderivatives of poly-N-acetylglucosamine polymers or fibers describedabove, can be irradiated when wet. The methods of making sNAG nanofibersby irradiation and the sNAG nanofibers so produced have been describedin U.S. Patent Pub. No. 2009/0117175, which is incorporated by referenceherein in its entirety.

In certain embodiments, the poly-N-acetylglucosamine polymers or fibersare formulated into a suspension/slurry or wet cake for irradiation.Irradiation can be performed prior to, concurrently with or followingthe formulation of the polymers or fibers into its final formulation,such as a dressing. Generally, the polymer or fiber content ofsuspensions/slurries and wet cakes can vary, for example from about 0.5mg to about 50 mg of polymer or fiber per 1 ml of distilled water areused for slurries and from about 50 mg to about 1000 mg of polymer orfiber per 1 ml of distilled water are use for wet cake formulations. Thepolymer or fiber may first be lyophilized, frozen in liquid nitrogen,and pulverized, to make it more susceptible to forming asuspension/slurry or wet cake. Also, the suspensions/slurries can befiltered to remove water such that a wet cake is formed. In certainaspects, the polymer or fiber is irradiated as a suspension comprisingabout 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10mg, 12 mg, 15 mg, 18 mg, 20 mg, 25 mg or 50 mg of polymer or fiber perml of distilled water, or any range in between the foregoing embodiments(e.g., 1-10 mg/ml, 5-15 mg/ml, 2-8 mg/ml, 20-50 mg/ml, etc.). In otheraspects, the polymer or fiber is irradiated as a wet cake, comprisingabout 50-1,000 mg polymer or fiber per 1 ml of distilled water. Inspecific embodiments, the wet cake comprises about 50, 100, 200, 300,400, 500, 600, 700, 800, 900 or 1000 mg of polymer or fiber per 1 mldistilled water, or any range in between (e.g., 100-500 mg/ml, 300-600mg/ml, 50-1000 mg/ml, etc.).

The irradiation is preferably in the form of gamma radiation, e-beamradiation, or x-rays. Two sources of irradiation are preferred:radioactive nuclides and electricity. In specific embodiment, theradioactive nuclides are cobalt-60 and cesium-137. Both of thesenuclides emit gamma rays, which are photons containing no mass. Thegamma rays have energies from 0.66 to 1.3 MeV. Using electricity,electrons are generated and accelerated to energies up to 10 MeV orhigher. When irradiating polymers or fibers to reduce their size, aconsideration to take into account is that the depth of penetration ofmaterials with densities similar to water by 10 MeV electrons is limitedto about 3.7 cm with one-sided exposure or about 8.6 cm with two-sidedexposure. Depth of penetration decreases at lower electron energies.Electron energy can be converted to x-rays by placing a metal (usuallytungsten or tantalum) target in the electron beam path. Conversion tox-rays is limited to electrons with energies up to 5 MeV. X-rays arephotons with no mass and can penetrate polymers or fibers similar togamma rays. There is only about 8% efficiency in the conversion ofelectron energy to x-ray energy. High powered electron beam machines areneeded in x-ray production facilities to account for the low conversionefficiency.

In a specific embodiment, the irradiation is gamma irradiation.

The absorbed dose of radiation is the energy absorbed per unit weight ofproduct, measured in gray (gy) or kilogray (kgy). For dried polymers orfibers, the preferred absorbed dose is about 500-2,000 kgy of radiation,most preferably about 750-1,250 kgy or about 900-1,100 kgy of radiation.For wet polymers or fibers, the preferred absorbed dose is about 100-500kgy of radiation, most preferably about 150-250 kgy or about 200-250 kgyof radiation.

The dose of radiation can be described in terms of its effect on thelength of the polymers or fibers. In specific embodiments, the dose ofradiation used preferably reduces the length of the polymer or fiber byanywhere from about 10% to 90% of the starting length of the polymer orfiber, respectively. In specific embodiments, the average length isreduced by about 10%, by about 20%, by about 30%, by about 40%, by about50%, by about 60%, by about 70%, by about 80%, or by about 90%, or anyrange in between (e.g., 20-40%, 30-70%, and so on and so forth).Alternatively, the dose of radiation used preferably reduces the lengthof the polymer or fiber to anywhere from 1 to 100 microns. In specificembodiments, and depending on the starting fiber length, the averagelength of the polymer or fiber is reduced to less than about 15 microns,less than about 14 microns, less than about 13 microns, less than about12 microns, less than about 11 microns, less than about 10 microns, lessthan about 8 microns, less than about 7 microns, less than about 5microns, less than about 4 microns, less than about 3 microns, less than2 microns, or less than 1 microns. In certain embodiments, the length ofthe majority (and in certain embodiments, at least 60%, 70%, 80%, 90%,95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or between 55% to 65%, 55%to 75%, 65% to 75%, 75% to 85%, 75% to 90%, 80% to 95%, 90% to 95%, or95% to 99%) of the polymers or fibers is reduced to no greater thanabout 20 microns, no greater than about 15 microns, no greater thanabout 12 microns, no greater than about 10 microns, no greater thanabout 8 microns, no greater than about 7 microns, or no greater thanabout 5 microns. In certain embodiments, irradiation of the polymers orfibers reduces the length of the majority (and in certain embodiments,at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or100%, or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the fibers to anywherebetween about 1 to 20 microns, between about 1 to 15 microns, betweenabout 2 to 15 microns, between about 1 to 12 microns, between about 2 to12 microns, between about 1 to 10 microns, between about 2 to 10microns, between about 1 to 8 microns, between about 2 to 8 microns,between about 1 to 7 microns, between about 2 to 7 microns, betweenabout 3 to 8 microns, between about 4 to 7 microns, between about 1 to 5microns, between about 2 to 5 microns, between about 3 to 5 microns,between about 4 to 10 microns, or any ranges between the foregoinglengths, which are also encompassed.

The dose of radiation can also be described in terms of its effect onthe molecular weight of the polymer or fiber. In specific embodiments,the dose of radiation used preferably reduces the molecular weight ofthe polymer or fiber by anywhere from about 10% to 90% of the startingweight of the polymer or fiber. In specific embodiments, the averagemolecular weight is reduced by about 10%, by about 20%, by about 30%, byabout 40%, by about 50%, by about 60%, by about 70%, by about 80%, or byabout 90%, or any range in between (e.g., 20-40%, 30-70%, and so on andso forth). Alternatively, the dose of radiation used preferably reducesthe molecular weight of the polymer or fiber to anywhere from 1,000 to1,000,000 daltons. In specific embodiments, and depending on thestarting molecular weight, the average molecular weight of the polymeror fiber is reduced to less than 1,000,000 daltons, less than 750,000daltons, less than 500,000 daltons, less than 300,000 daltons, less than200,000 daltons, less than 100,000 daltons, less than 90,000 daltons,less than 80,000 daltons, less than 70,000 daltons, less than 60,000daltons, less than 50,000 daltons, less than 25,000 daltons, less than10,000 daltons, or less than 5,000 daltons. In certain embodiments, theaverage molecular weight is reduced to no less than 500 daltons, no lessthan 1,000 daltons, no less than 2,000 daltons, no less 3,500 daltons,no less than 5,000 daltons, no less than 7,500 daltons, no less than10,000 daltons, no less than 25,000 daltons, no less than 50,000daltons, no less than 60,000 daltons or no less than 100,000 daltons.Any ranges between the foregoing average molecular weights are alsoencompassed; for example, in certain embodiments, irradiation of thepolymer or fiber reduces the average molecular weight to anywherebetween 10,000 to 100,000 daltons, between 1,000 and 25,000 daltons,between 50,000 and 500,000 daltons, between 25,000 and 100,000 daltons,between 30,000 and 90,000 daltons, between about 40,000 and 80,000daltons, between about 25,000 and 75,000 daltons, between about 50,000and 70,000 daltons, or between about 55,000 and 65,000 daltons and so onand so forth. In certain embodiments, irradiation of the polymers orfibers reduces the molecular weight of the majority and in certainembodiments, at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%,99.9%, or 100%, or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to85%, 75% to 90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the fibers toanywhere between about 20,000 and 100,000 daltons, about 25,000 and75,000 daltons, about 30,000 and 90,000 daltons, about 40,000 and 80,000daltons, about 50,000 and 70,000 daltons, or about 55,000 and 65,000daltons. In certain embodiments, irradiation of the polymers or fibersreduces the molecular weight of the majority and in certain embodiments,at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or100%, or between 55% to 65%, 55% to 75%, 65% to 75%, 75% to 85%, 75% to90%, 80% to 95%, 90% to 95%, or 95% to 99%) of the fibers to about60,000 daltons.

Following irradiation, slurries can be filtered and dried, and wet cakescan be dried, to form compositions (e.g., dressings and othercompositions described herein) that are useful in the practice of theinvention.

5.3 Compositions Comprising sNAG Nanofibers

The sNAG nanofibers may be formulated in a variety of compositions fortopical administration as described herein.

A composition comprising the sNAG nanofibers may be formulated as acream, a membrane, a film, a liquid solution, a suspension, a powder, apaste, an ointment, a suppository, a gelatinous composition, an aerosol,a gel, or a spray. In one embodiment, a composition comprising the sNAGnanofibers is formulated as an ultra-thin membrane. In some embodiments,a composition comprising the sNAG nanofibers is formulated as adressing, a mat, or a bandage. Solid formulations suitable for solutionin, or suspension in, liquids prior to administration are alsocontemplated. It is also possible that such compositions areincorporated in or coated on implantable devices, such as orthopedicimplants (for hip, knee, shoulder; pins, screws, etc.), cardiovascularimplants (stents, catheters, etc.) and the like where the antibacterialactivity would be of benefit.

A composition comprising the sNAG nanofibers may include one or more ofpharmaceutically acceptable excipients. Suitable excipients may includewater, saline, salt solution, dextrose, glycerol, ethanol and the like,or combinations thereof. Suitable excipients also include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, oil (including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like), talc, sodiumchloride, dried skim milk, propylene, glycol and the like. In addition,a composition comprising the sNAG nanofibers may include one or more ofwetting agents, emulsifying agents, pH buffering agents, and otheragents. The sNAG nanofiber compositions may also be incorporated in aphysiologically acceptable carrier, for example in a physiologicallyacceptable carrier suitable for topical application. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The final amount of the sNAG nanofibers in a composition may vary. Forexample, the amount of the sNAG nanofibers in a composition (e.g.,prepared for administration to a patient) may be greater than or equalto about 50%, about 60%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 98%, or about 99% weight by volume. In oneembodiment, the amount of the sNAG nanofibers in a composition is about95%, about 98%, about 99, or about 100%. Also, the amount of the sNAGnanofibers in a composition (e.g., prepared for administration to apatient) may be about 50%-100%, about 60%-100%, about 70%-100%, about75%-100%, about 80%-100%, about 90%-100%, about 95%-100%, about 70%-95%,about 75%-95%, about 80%-95%, about 90%-95%, about 70%-90%, about75%-90%, or about 80%-90% weight/volume. A composition may comprise morethan 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or 99% solution of thesNAG nanofibers.

A sNAG nanofiber composition may be formulated into a wound dressing. Incertain embodiments, a sNAG nanofiber composition is formulated as awound dressing in the form of a barrier, a membrane, or a film.Alternatively, a sNAG nanofiber composition may be added to dressingbackings, such as barriers, membranes, or films. A barrier, membrane, orfilm can be supplied in a variety of standard sizes, which can befurther cut and sized to the area being treated. The backing can be aconventional dressing material, such as a bandage or gauze to which apolymer or fiber is added or coated on, prior to application to thepatient. Alternatively, the sNAG nanofibers can be formulated as abarrier, membrane, or film made out of strings, microbeads,microspheres, or microfibrils, or the composition can be formulated as abarrier-forming mat. In certain embodiments, at least 75%, at least 85%,at least 90%, or at least 95% of a dressing is composed of the sNAGnanofibers. In certain aspects, a dressing does not contain aconventional dressing material such as a gauze or bandage. In suchembodiments, the sNAG nanofiber itself is formulated as a wounddressing.

A composition comprising the sNAG nanofibers may further comprise anysuitable natural or synthetic polymers or fibers. Examples of suitablepolymers or fibers include cellulose polymers, xanthan, polyaramides,polyamides, polyimides, polyamide/imides, polyamidehydrazides,polyhydrazides, polyimidazoles, polybenzoxazoles, polyester/amide,polyester/imide, polycarbonate/amides, polycarbonate/imides,polysulfone/amides, polysulfone imides, and the like, copolymers andblends thereof. Other suitable classes of polymers or fibers includepolyvinyledene fluorides and polyacrylonitriles. Examples of thesepolymers or fibers include those described in U.S. Pat. Nos. RE 30,351;4,705,540, 4,717,393; 4,717,394; 4,912,197; 4,838,900; 4,935,490;4,851,505; 4,880,442; 4,863,496; 4,961,539; and European PatentApplication 0 219 878, all of which are incorporated by reference. Thepolymers or fibers can include at least one of either of cellulosepolymers, polyamides, polyaramides, polyamide/imides or polyimides. Incertain embodiments, the polymers or fibers include polyaramides,polyester, urethan and polytetrafluoroethylene. In one embodiment, thecompositions described herein comprise more than one type of polymer(e.g., the sNAG nanofiber and cellulose).

In certain aspects, the sNAG nanofiber is the only active ingredient ina composition.

In other embodiments, a composition comprises one or more additionalactive ingredients, e.g., to promote an anti-bacterial effect and/orhealing (e.g., wound healing). In some embodiments, the additionalactive ingredient is one or more anti-bacterial agents (e.g., anantibiotic, a defensin peptide, a defensin-like peptide, or aToll-receptor-like peptide), or a growth factor. In specificembodiments, the additional active ingredient is a growth factor such asone or more of PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, FGF-1,FGF-2, FGF-5, FGF-7, FGF-10, EGF, TGF-α, (HB-EGF), amphiregulin,epiregulin, betacellulin, neuregulins, epigen, VEGF-A, VEGF-B, VEGF-C,VEGF-D, VEGF-E, placenta growth factor (PLGF), angiopoietin-1,angiopoietin-2, IGF-I, IGF-II, hepatocyte growth factor (HGF), andmacrophage-stimulating protein (MSP). In other embodiments, theadditional active ingredient is an agent that boost the immune system, apain relief agent, or a fever relief agent.

In certain embodiments, the additional active ingredient is anantibiotic of one of the following classes of antibiotics: microlides(e.g., erythromycin, azithromycin), aminoglycosides (e.g., amikacin,gentamicin, neomycin, streptomycin), cephalosporins (e.g., cefadroxil,cefaclor, cefotaxime, cefepime), fluoroquinolones (e.g., ciprofloxacin,levofloxacin), penicillins (e.g., penicillin, ampicillin, amoxicillin),tetracyclines (e.g., tetracycline, doxycycline), and carbapenems (e.g.,meropenem, imipenem). In some specific embodiments, the additionalactive ingredient is one or more of vancomycin, sulfa drug (e.g.,co-trimoxazole/trimethoprim-sulfamethoxazole), tetracycline (e.g.,doxycycline, minocycline), clindamycin, oxazolidinones (e.g.,linezolid), daptomycin, teicoplanin, quinupristin/dalfopristin(synercid), tigecycline, allicin, bacitracin, nitrofurantoin, hydrogenperoxide, novobiocin, netilmicin, methylglyoxal, bee defensin-1,tobramycin, chlorhexidine digluconate, chlorhexidine gluconate,levofloxacin, zinc, and silver. In some embodiments, a compositioncomprises the sNAG nanofibers and an additional active ingredienteffective to treat or prevent or commonly used to treat or prevent an S.aures infection, MRSA infection, a Pseudomonas infection, or a C.dificule infection (e.g., an antibiotic effective against or commonlyused against such infections).

A sNAG nanofiber composition may contain collagen, although in certainaspects a sNAG nanofiber composition does not contain collagen.

In certain embodiments, a sNAG nanofiber composition does not compriseany additional therapy. In certain embodiments, a sNAG nanofibercomposition does not comprise any additional anti-bacterial agent, adefensin peptide, a defensin-like peptide, a Toll-receptor-like peptide,or a growth factor. In some embodiments, a sNAG nanofiber compositiondoes not comprise an antibiotic. In yet other embodiments, a sNAGnanofiber composition may comprise an additional therapy (e.g., anantibiotic). In one such embodiment, the additional therapy (e.g., anantibiotic) is not encapsulated, immobilized or formulated in the sNAGnanofibers.

In other aspects, a sNAG nanofiber composition does not comprise asignificant amount of protein material. In specific embodiments, theprotein content of a sNAG nanofiber composition is no greater than 0.1%,0.5% or 1% by weight. In other embodiments, the protein content of thecomposition is undetectable by Coomassie staining.

In one embodiment, zinc is also included in a sNAG nanofibercomposition. In addition to its antimicrobial properties, zinc alsoplays a role in wound healing (see Andrews et al., 1999, Adv Wound Care12:137-8). The zinc is preferably added in the form of a salt, such aszinc oxide, zinc sulphate, zinc acetate or zinc gluconate.

5.4 Anti-Bacterial Uses of sNAG Compositions

A wide variety of bacterial infections and diseases associated therewithmay be treated and/or prevented by the administration of the sNAGnanofiber compositions described herein (see, e.g., Sections 5.4.1 and5.4.2, infra). In one embodiment the compositions described herein arebacteriostatic. In another embodiment, the compositions described hereinare bactericidal. In an embodiment, the compositions described hereinmay be used to treat and/or prevent infections by Gram-positive bacteriaand/or any diseases associated therewith. In another embodiment, thecompositions described herein may be used to treat and/or preventinfections by Gram-negative bacteria and/or any diseases associatedtherewith. In yet another embodiment, the compositions described hereinmay be used to treat and/or prevent infections by both Gram-negativebacteria and Gram-positive bacteria and/or any diseases associatedtherewith.

Bacterial infections that may be treated and/or prevented usingcompositions described herein include infections by bacteria of theAquaspirillum family, Azospirillum family, Azotobacteraceae family,Bacteroidaceae family, Bartonella species, Bdellovibrio family,Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae),clostridium, Enterobacteriaceae family (e.g., Citrobacter species,Edwardsiella, Enterobacter aerogenes, Erwinia species, Escherichia coli,Hafnia species, Klebsiella species, Morganella species, Proteusvulgaris, Providencia, Salmonella species, Serratia niarcescens, andShigella flexneri), Gardinella family, Haemophilus influenzae,Halobacteriaceae family, Helicobacter family, Legionallaceae family.Listeria species, Methylococcaceae family, mycobacteria (e.g.,Mycobacterium tuberculosis), Neisseriaceae family, Oceanospirillumfamily, Pasteurellaceae family, Pneumococcus species, Pseudomonasspecies, Rhizobiaceae family, Spirillum family, Spirosomaceae family,Staphylococcus (e.g., methicillin resistant Staphylococcus aureus andStaphylococcus pyrogenes), Streptococcus (e.g., Streptococcusenteritidis, Streptococcus fasciae, and Streptococcus pneumoniae),Vampirovibr Helicobacter family, and/or Vampirovibrio family. In aspecific embodiment, diseases caused by or associated with infections bysuch bacteria may also be prevented and/or treated using thecompositions described herein.

Bacterial infections that may be treated and/or prevented usingcompositions described herein also include infections by bacteria of thefollowing genuses: Bordetella, Borrelia, Brucella, Campylobacter,Chlamydia and Clamidophylia, Clostridium, Corynebacterium, Enterococcus,Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Treponema, Vibria, and/or Yersinia. In a specific embodiment, diseasescaused by or associated with infections by such bacteria may also beprevented and/or treated using the compositions described herein.

Bacterial infections that may be treated and/or prevented usingcompositions described herein include infections by bacteria of thefollowing species: Bacillus anthracis, Bordetella pertussis, Borreliaburgdorferi, Brucella abortus, Brucella canis, Brucella melitensis,Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydiatrachomatis, Clamidophila psittaci, Clostridium botulinum, Clostridiumdificule, Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenae, Helicobacterpylori, Legionella pneumphila, Leptospira pneumophila, Leptospirainterrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitides, Pseudomonas aeruginosa, Proteus mirabilis, Rickettsiarickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumonia,Streptococcus pyogenes, Treponema pallidum, Vibria cholerae, and/orYersinia pestis. In a specific embodiment, diseases caused by orassociated with infections by such bacteria may also be prevented and/ortreated using the compositions described herein.

In some embodiments, the compositions described herein may be used totreat and/or prevent infections by aerobic bacteria and/or diseasesassociated therewith. In other embodiments, the compositions describedherein may be used to treat and/or prevent infections by anaerobicbacteria and/or diseases associated therewith.

In certain embodiments, the compositions described herein are used totreat and/or prevent Pseudomonas aeruginosa infections. Pseudomonas is agram-negative aerobic bacteria found in soil, water, other moistenvironments, plants and animals, clinical isolates of which produce theblue-green pigment pyocyanin and a characteristic sweet odor.Pseudomonas aeruginosa is known to cause urinary tract infections,pneumonia, respiratory system infections, dermatitis, soft tissueinfections, bacterimia, bone and joint infections, gastrointestinalinfections and a variety of systemic infections. It is known to be animportant cause of infections, particularly in patients with burns,patients with cystic fibrosis, patients who are immunosuppressed (e.g.,AIDS and cancer patients), and in patients who have been hospitalizedfor longer than 1 week. It is a frequent cause of nosocomial infectionssuch as but not limited to pneumonia, urinary tract infections andbacterimia. Any one or all of these infections may be prevented and/ortreated by the compositions described herein.

In some embodiments, the compositions described herein are used to treatand/or prevent Staph infections, and particularly, Staphylococcus aureusinfections. Use of a sNAG nanofiber composition in this embodiment andother embodiments described herein may preclude the generation ofresistant organisms as well as allow for the antibiotic-independentclearance of a bacterial infection.

In certain embodiments, the compositions described herein may be used tocombat bacteria that are resistant to one or more anti-bacterial agents.For example, the compositions described herein may be used to treatbacteria that are resistant to one or more antibiotics, for exampleresistant to conventional antibiotics such as MRSA(methicillin-resistant Staphylococcus aureus), VRSA(Vancomycin-resistant S. aureus), VRE (Vancomycin-resistantEnterococus), Penicillin-resistant Enterococcus, PRSP(Penicillin-resistant Streptococcus pneumonia),isoniazid/rifampin-resistant Mycobacterium tuberculosis and otherantibiotic-resistant strains of bacteria (e.g., resistant strains of E.coli, Salmonella, Campylobacter, and Streptococci). In one embodiment,the compositions disclosed herein may be used to treat multiple drugresistant bacteria.

In some specific embodiments, the compositions described herein may beused to treat and/or prevent Methicillin-resistant Staphylococcus aureus(“MRSA”; it may also be called multidrug-resistant Staphylococcus aureusor oxacillin-resistant Staphylococcus aureus (“ORSA”)). MRSA is anystrain of Staphylococcus aureus that has developed resistance tobeta-lactam antibiotics, which include but are not limited to thepenicillins (penicillin, methicillin, dicloxacillin, nafcillin,oxacillin, etc.) and the cephasosporins. Some of the known strains ofMRSA include EMRSA15 and EMRSA16 (also known as MRSA252), which areresistant to erythromycin and ciprofloxacin; CC8 (also known asST8:USA300); ST1:USA400; ST8:USA500; ST59:USA000; ST93 strains; ST80strains; and ST59 strains. MRSA is responsible for a number ofinfections in humans. MRSA is a serious health concern, causingapproximately 50% of health-care associated staph infections. In theU.S., more than 94,000 people develop serious MRSA infection and about19,000 die from infection each year. Especially prevalent MRSA is inhospitals; where risk factors for MRSA infection include priorantibiotic exposure (e.g., quinolone antibiotics), admission to anintensive care unit, surgery and exposure to an MRSA-colonized patient.Patients with open wounds, immunocompromised patients (due to, e.g.,HIV/AIDS, cancer, transplant procedure, severe asthma), young children(e.g., human infant and human toddler), and the elderly (e.g., elderlyhuman) are at high risk of developing an MRSA infection. Higher riskrates for MRSA infection are also observed in injection drug users,persons with diabetes, patients with dermatologic conditions, patientswith invasive devices (e.g., intravascular catheters), and health careworkers and other people who spend time in confined spaces (prisoninmates, soldiers, patients in long-term healthcare facilities, such asnursing homes). S. aureus most commonly colonizes the anterior nares(the nostrils), although the rest of the respiratory tract, openedwounds, intravenous catheters and urinary tract are also potential sitesfor infection. Most of community-associated MRSA infections arelocalized to the skin and soft tissue. The initial symptoms of MRSAinclude red bumps that resemble pimples, spider bites or boils that maybe accompanied by fever and rashes; the bumps may later develop intopus-filled boils. Common manifestations of community-associated MRSA areskin infections such as necrotizing fasciitis or pyomyositis,necrotizing pneumonia, infective endocarditis, bone or joint infections.Some MRSA leads to sepsis and toxic shock syndrome, which may be due totoxins carried by such strains (e.g., PVL, PSM). MRSA may causecellulitis. Any of the above-listed or known in the art strains of MRSA,patients diagnosed with MRSA, symptoms of MRSA, patient populations atrisk of MRSA and/or diseases associated with MRSA may be treated withthe compositions described herein. In some embodiments, the compositionsdescribed herein prevent onset or development of one or more of thesymptoms of MRSA, or reduce duration and/or severity of one or more ofthese symptoms (e.g., symptoms described herein).

The compositions described herein may be used as bactericidal agents tokill or damage unwanted bacteria. For example, the compositionsdescribed herein may be used to treat established bacterial infections,prophylactically for the prevention of bacterial infections, oradministered topically to areas of a subject that are susceptible toinfection or to areas of the body that are likely sites for bacterialgrowth (e.g., the gums, open wounds, bed sores, and vaginal or groinareas).

In certain embodiments, the compositions described herein reducebacterial growth and/or bacterial survival by more than about 0.1 log,0.2 log, 0.25 log, 0.3 log, 0.4 log, 0.5 log, 0.6 log, 0.7 log, 0.75log, 0.8 log, 0.9 log, 1 log, 1.25 log, 1.5 log, 1.75 log, 2 log, 2.25log, 2.5 log, 2.75 log, 3 log, 3.25 log, 3.5 log, 3.75 log, 4 log, 4.5log, 5 log, 5.5 log, 6 log, 6.5 log, 7 log, 7.5 log, 8 log, 8.5 log, 9log, 9.5 log, 10 log, 10.5 log, 11 log, 11.5 log, 12 log, 12.5 log, 13log, 13.5 log, 14 log, 14.5 log, or 15 log of colony forming units(CFU)/mL. In certain embodiments, the compositions described hereinreduce bacterial growth and/or bacterial survival by about 0.2 log to 15log, 0.2 log to 10 log, 0.2 log to 5 log, 0.5 log to 15 log, 0.5 log to10 log, 0.5 log to 5 log, 0.5 log to 3 log, 1 log to 15 log, 1 log to 12log, 1 log to 10 log, 1 log to 7 log, 1 log to 5 log, 1 log to 3 log,1.5 log to 5 log, 2 log to 15 log, 2 log to 10 log, 2 log to 5 log, 3log to 15 log, 3 log to 10 log, 3 log to 5 log, 4 log to 10 log, 2 logto 8 log, 3 log to 8 log, 4 log to 8 log, 2 log to 7 log, 3 log to 7log, 2 log to 6 log of colony forming units (CFU)/mL, and any value inbetween these values. In certain embodiments, the compositions describedherein reduce bacterial growth and/or bacterial survival by equal to ormore than 1×10¹⁰, 0.5×10¹¹, 1×10¹¹, 1.5×10¹¹, 2×10¹¹, 2.5×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 7×10¹¹, 1×10¹², 1.5×10¹², 2×10¹², 3×10¹², 5×10¹²,7×10¹², 8×10¹², 1×10¹³, 1.5×10¹³, or 2×10¹³ (CFU)/mL, or any range ofvalues in between these values. In some embodiments, such reduction inbacterial growth and/or survival is achieved in less than about 30minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 20hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, oneday, two days, three days, four days, five days, seven days, ten days,one week, two weeks, three weeks, four weeks, 1 month or 2 months aftertreatment of a bacterial infection with a single application/dose ormultiple application/doses of a sNAG nanofiber composition.

In one embodiment, the infection to be treated with a sNAG nanofibercomposition is not a viral infection, a fungal infection, a parasiteinfection, or an yeast infection.

A variety of diseases or disease conditions associated with bacterialinfections may be treated and/or prevented with the sNAG nanofibercompositions described herein (see, e.g., Section 5.4.2, infra). In oneembodiment, methods for treating an existing bacterial infection or adisease associated with a bacterial infection are contemplated.

In some embodiments, the compositions described herein may be used totreat wounds (see Section 5.4.1, infra). In specific embodiments, thecompositions described herein are used to treat bacterially infectedwounds. In other embodiments, the compositions described herein are usedto prevent bacterial infection of wounds. In some embodiments, thecompositions described herein are used to treat bacteria known to beassociated with wound infections, and specifically used to treatStaphylococcus aureus/MRSA, Streptococcus pyrogenes, Enterococci and/orPseudomonas aeuruginosa infections, and diseases associated with suchinfections.

In other embodiments, the compositions described herein are not used totreat wounds. In one embodiment, the compositions described herein arenot used to treat chronic wounds. In another embodiment, thecompositions described herein are not used to treat burn wounds. In yetanother embodiment, the compositions described herein are not used totreat surgical wounds. In one embodiment, the compositions describedherein are not used to treat chronic wounds, burn wounds and surgicalwounds. In another embodiment, the compositions described herein are notused to treat a wound and/or a burn. In yet another embodiment, thecompositions described herein are not used to treat un-infected wounds.

In some embodiments, the compositions described herein are not used totreat a bacterially infected wound. In another embodiment, thecompositions described herein are not used to treat a bacterialinfection associated with or caused by a wound. In some embodiments, thecompositions described herein are not used to treat bacteria known to beassociated with wound infections such as Staphylococcus aureus/MRSA,Streptococcus pyrogenes, Enterococci and/or Pseudomonas aeuruginosa.

In some embodiment, the compositions described herein may be used totreat or prevent a variety of bacterial infections and diseases causedby or associated with bacterial infections, which are not associatedwith a wound (see Section 5.4.2, infra).

In certain embodiments, treatment of a disease associated with abacterial infection comprises administration of one of the compositionsdescribed herein to a subject or a population of subjects to treat thedisease or to obtain a beneficial or therapeutic effect. In specificembodiments, such treatment achieves one, two, three, four, five or moreof the following effects in a subject or a population of subjects: (i)reduction or amelioration of the severity of a disease or a symptomassociated therewith; (ii) reduction of the duration of a disease or asymptom associated therewith; (iii) prevention of the progression of adisease or a symptom associated therewith; (iv) regression of a diseaseor a symptom associated therewith; (v) prevention of the development oronset of a symptom associated therewith; (vi) prevention of therecurrence of a symptom associated therewith; (vii) prevention orreduction of the spread of a disease from the subject or population ofsubjects to another subject or population of subjects; (viii) reductionin organ failure associated with a disease; (ix) reduction of theincidence of hospitalization; (x) reduction of the hospitalizationlength; (xi) an increase the survival; (xii) elimination of a disease;(xiii) enhancement or improvement of the prophylactic or therapeuticeffect(s) of another therapy; (xiv) improvement in quality of life asassessed by methods well known in the art, e.g., a questionnaire; (xv)reduction of the number of symptoms of a disease; and/or (xvi) reductionin mortality. In some embodiments, treatment comprises any therapy usingcompositions described herein.

In some embodiments treatment of a bacterial infection comprisesadministration of one of the compositions described herein to a subjector a population of subjects to treat the bacterial infection or asymptom of a bacterial infection. In specific embodiments, suchtreatment achieves one, two, three, four, five or more of the followingeffects in a subject or a population of subjects: (i) the clearance of abacterial infection; (ii) the eradication of one or more symptomsassociated with a bacterial infection, (iii) the reduction of timerequired to clear a bacterial infection; (iv) the reduction oramelioration of the severity of a bacterial infection and/or one or moresymptoms associated therewith; (v) the reduction in the duration of abacterial infection and/or one or more symptoms associated therewith;(vi) the prevention or delay of the generation of a resistant strain orstrains of bacteria or reduction of a number of resistant strains ofbacteria generated; (vii) the prevention in the recurrence of one ormore symptoms associated therewith; (viii) the reduction or eliminationin the bacterial cell population (such as reduction in bacterial counts,e.g., in a biological sample of a patient, as measured by CFU/mL or alog reduction by one of the methods known in the art or describedherein); (ix) the reduction in hospitalization of a subject; (x) thereduction in hospitalization length; (xi) the increase in the survivalof a subject; (xii) the enhancement or improvement of the therapeuticeffect of another therapy; (xiii) a reduction in mortality; (xiv) thereduction or elimination in the spread of the bacteria from one subjectto another subject, or one organ or tissue to another organ or tissue;(xv) the prevention of an increase in the number of bacteria; (xvi) theprevention of the development or onset of one or more symptomsassociated therewith; (xvii) the reduction in the number of symptomsassociated with a bacterial infection; (xviii) the inhibition orreduction in production of a bacterial toxin or toxins associated with abacterial infection; (xix) the stabilization or reduction ofinflammation associated with a bacterial infection; (xx) the reductionin organ failure associated with a bacterial infection or a diseaseassociated therewith; and/or (xxi) improvement in quality of life asassessed by methods well known in the art, e.g., a questionnaire.

In certain embodiments, administration of the compositions describedherein to a subject results in one or more of the following: (i) theinduction of the expression of one or more defensin proteins and/ordefensin-like proteins; (ii) the induction of the expression of one ormore Toll-like receptors; and/or (iii) the induction of the expressionof one or more proteins that are beneficial for clearance or reductionof a bacterial infection or one or more symptoms associated therewith.

In certain embodiments, prevention of a bacterial infection comprisesadministration of one of the compositions described herein to a subjector a population of subjects to achieve one or more of the followingeffects: (i) the inhibition of the development or onset of a bacterialinfection, or a symptom associated therewith; and/or (ii) the inhibitionof the recurrence of a bacterial infection, or a symptom associatedtherewith.

In other embodiments, prevention of a bacterial infection comprisesadministration of one of the compositions described herein to a subjector a population of subjects to prevent a disease associated with abacterial infection. In specific embodiments, such prevention achievesone or more of the following effects in a subject or a population ofsubjects: (i) the inhibition of the development or onset of a diseaseassociated with a bacterial infection or a symptom thereof; and/or (ii)the inhibition of the recurrence of a disease associated with abacterial infection or a symptom associated therewith.

5.4.1 Treatment or Prevention of Bacterial Infection in Wounds

In certain embodiments, the sNAG nanofiber compositions described hereinmay be useful for treating a wide variety of bacterially infected woundsaffecting any tissue of the body or preventing infection of wounds atrisk of becoming infected with bacteria.

There are two types of wounds, open and closed. Open wounds areclassified according to the object that caused the wound. For example,incisions or incised wounds (including surgical wounds) are caused by aclean, sharp-edged object such as a knife, a razor or a glass splinter.Lacerations are irregular wounds caused by a blunt impact to soft tissuewhich lies over hard tissue (e.g., laceration of the skin covering theskull) or tearing of skin and other tissues such as caused bychildbirth. Abrasions or grazes are superficial wounds in which thetopmost layer of the skin (the epidermis) is scraped off. Puncturewounds are caused by an object puncturing the skin, such as a nail orneedle. Penetration wounds are caused by an object such as a knifeentering the body. Gunshot wounds are caused by a bullet or similarprojectile driving into (e.g., entry wound) and/or through the body(e.g., exit wound). In a medical context, all stab wounds and gunshotwounds are considered open wounds. Open wounds also include burn woundsinduced by thermal, chemical, or electrical injury. Closed woundsinclude contusions (more commonly known as a bruise, caused by bluntforce trauma that damages tissue under the skin), hematoma (also calleda blood tumor, caused by damage to a blood vessel that in turn causesblood to collect under the skin), and crushing injuries (caused by agreat or extreme amount of force applied over a long period of time).

In certain embodiments, the compositions described herein are used totreat a bacterial infected open wound or prevent a bacterial infectionin an open wound. In certain embodiments, the compositions describedherein may be used to treat or prevent a bacterial infection of agunshot wound, a puncture wound and/or a penetration wound. In certainembodiments, the compositions described herein may be used to treat orprevent a post-operative bacterial infection, a surgical site bacterialinfection, a catheter-related bacterial infection or ahemodialysis-related bacterial infection. In yet another embodiment, thecompositions described herein are not used to treat or prevent abacterial infection in an open wound, a gunshot wound, a puncture woundand/or a penetration wound. In certain embodiments, the compositionsdescribed herein are not used to treat or prevent a post-operativebacterial infection, a surgical site bacterial infection, acatheter-related bacterial infection or hemodialysis-related bacterialinfection.

In some embodiments, the wound is a chronic wound. Chronic wound can beany wound that fails to heal properly, including a surgical wound (e.g.,a skin graft donor site), a cutaneous ulcer (e.g., a diabetic ulcer, avenous stasis ulcer, a leg ulcer, an arterial insufficiency ulcer, or apressure ulcer), or a burn wound. In one embodiment, the compositionsdescribed herein are used to treat or prevent chronic wound infections(e.g. an infection associated with a diabetic ulcer, a venous stasisulcer, a leg ulcer, an arterial insufficiency ulcer, a pressure ulcer, asurgical wound, or a burn). In yet another embodiment, the compositionsdescribed herein are not used to treat or prevent chronic woundbacterial infections (e.g. not used to prevent a bacterial infectionassociated with a diabetic ulcer, a venous stasis ulcer, a leg ulcer, anarterial insufficiency ulcer, a pressure ulcer, a surgical wound, or aburn).

In certain embodiments, the compositions described herein are used totreat or prevent nosocomial bacterial infections. Of the nosocomialbacterial infections, surgical wound bacterial infections predominate;with statistics showing up to 8% of all surgical patients. The directcost of these types of infections is approximately 4.5 billion dollarsper year. Many of hospital-contracted bacteria developed resistanceagainst antibiotics, and thus non-antibiotic-based treatments aredesired. Use of the sNAG compositions described herein in a hospitalsetting could defray much of the cost and markedly reduce the productionof antibiotic resistant species. In yet another embodiment, thecompositions described herein are not used to treat or preventnosocomial bacterial infections, such as surgical bacterial infections.

In one embodiment, the compositions described herein may be used totreat or prevent bacterial infections in bleeding wounds (e.g., bleedingsurface wounds). In one embodiment, the compositions described hereinmay be used to treat a gunshot wound, a puncture wound, a penetrationwound or a surgical wound in order to treat or prevent bacterialinfection in such wound. In yet another embodiment, the compositionsdescribed herein are not used to treat or prevent bacterial infectionsin bleeding wounds (e.g., bleeding surface wounds).

The compositions described herein may be useful for treating orpreventing a bacterial infection in cutaneous wounds, such as woundsaffecting the epidermal and dermal layers of the skin, as well asinjuries to the cornea and epithelia-lined organs, in order to treat orprevent a bacterial infection in such wounds. The wounds may be causedby a wide variety of physical trauma, including cuts, abrasions, burns,chemical exposure, surgical procedures (e.g., surgical incisions, skingrafting). In one embodiment, the compositions described herein may beused for treating corneal and sclera wounds, including wounds whichaffect the epithelial layer, stromal layer and endothelial layers of theeye in order to treat or prevent a bacterial infection in such wounds.In yet another embodiment, the compositions described herein are notused to treat or prevent bacterial infections in cutaneous wounds.

In some embodiments, the compositions described herein may be used totreat a wound in a patient diagnosed with a bacterial infection. Incertain embodiments, where the compositions described herein are used totreat a bacterial infected wound, a wound is determined to bebacterially infected by a test or an assay for the presence of abacterial antigen. In one embodiment, a wound culture is performed todetect a bacterial infection in the wound of a patient. In yet otherembodiments, a wound is determined to be infected due to the presence ofone or more symptoms of bacterial infection.

In other embodiments, the compositions described herein may be used totreat a wound in a patient when a patient displays one or more of thesymptoms of bacterial infection such as: a wound is slow to heal; heat,redness and/or swelling at the site of the wound; tenderness at the siteof the wound; drainage of fluid or pus at the site of the wound; and/orfever. Symptoms of wound bacterial infection include but are not limitedto localized erythema, localized pain, localized heat, cellulitis,oedema, abscess, discharge which may be viscous, discolored andpurulent, delayed of wound healing, discoloration of tissues both withinand/or at the wound margins, friable, bleeding granulation tissue,abnormal smell coming from the wound site, unexpected pain and/ortenderness at the site of dressing change, lymphangitis (i.e., a redline originating from the wound and leading to swollen tender lymphglands draining the affected area), and wound breakdown associated withwound pocketing/bridging at base of wound (i.e., a wound develops stripsof granulation tissue in the base as opposed to a uniform spread ofgranulation tissue across the whole of the wound bed). In someembodiments, the compositions described herein prevent the onset ordevelopment of one or more of the above-listed symptoms, or reduceduration and/or severity of one or more of these symptoms.

In one embodiment, the compositions described herein may be used forwound healing and treatment of a wound bacterial infection, or for woundhealing and prevention of a wound bacterial infection. In oneembodiment, the compositions described herein are used to enhance woundhealing while concurrently treating or preventing a wound bacterialinfection. Effects of the sNAG nanofiber compositions on wound healingand some of the uses of the sNAG nanofibers in wound healingapplications have been described in U.S. Patent Pub. No. 2009/0117175,which is incorporated by reference herein in its entirety (see, e.g.,Example 2).

5.4.2 Treatment or Prevention of Other Bacterial Infections

In certain embodiments, the sNAG nanofiber compositions described hereinmay be used to treat and/or prevent bacterial infections of the skin,gastrointestinal tract, respiratory tract, urinary tract, reproductivetract, blood, throat, ears, eye, sinus or any other organ or tissue ofthe body. In another embodiment, the sNAG nanofiber compositionsdescribed herein may be used to treat and/or prevent skin conditions,gastrointestinal conditions, respiratory conditions and/or conditions ofany other organ or tissue associated with a bacterial infection. In someembodiments, the sNAG nanofiber compositions described herein areapplied topically on the skin, mouth, ear, eye, anus or groin areas of apatient to treat or prevent a bacterial infection. In some embodiments,the compositions described herein are used to treat and/or prevent abacterial infection of an organ or tissue of the body that is not at thesite of a wound, and/or is not associated with or caused by a wound.

In certain embodiments, the sNAG nanofiber compositions described hereinare used to treat an existing bacterial infection. For example, suchcompositions may be used to treat a subject diagnosed with a bacterialinfection by a test or an assay, such as one of the tests describedherein or known in the art. Alternatively, such compositions may be usedto treat a subject displaying one or more symptoms of a bacterialinfection or a disease associated with a bacterial infection, such asone or more symptoms of a bacterial infection known to a skilled artisan(e.g., determined by a treating doctor/physician to be a symptom of abacterial infection) and/or described herein.

In certain embodiments, the compositions described herein are used totreat a condition associated with an imbalance in bacterial microbiota,or a condition associated with an abnormal or altered bacterialmicrobiota. For example, such compositions may be used to treat a skincondition in a patient whose skin bacterial microbiota differs from thatin control subjects (e.g., subjects with no symptoms of the skincondition). In other examples, such compositions may be used to treat anintestinal condition (or a condition of any other tissue or organ) in apatient whose intestinal bacterial microbiota (or microbiota of theother tissue or organ) differs from that in control subjects (e.g.,subjects with no symptoms of the intestinal condition).

In other embodiments, the compositions described herein may be used totreat any disease known to be associated with or exacerbated by abacterial infection (e.g., acne). In one embodiment, the compositionsdescribed herein may be used to treat a cystic fibrosis patient infectedby P. aeruginosa.

In some embodiments, the compositions described herein are effectiveagainst toxins secreted or excreted by bacteria. In one embodiment, thecompositions described herein may be used to inhibit/reduce a bacterialtoxin (and/or a condition or symptom caused by a bacterial toxin), forexample toxins produced by Bacillus anthracis, Clostridium difficile,Corvnebacterium diphtheria, Pseudomonas aeruginosa; endotoxins, and/orthe cytolysins. Some defensins are able to inhibit bacterial toxins,including those produced by Bacillus anthracis, Clostridium difficile,Cornebacterium diphtheria, Pseudomonas aeruginosa, and the cytolysins(endotoxins produced by Gram-positive bacteria that lyse red bloodcells). Given these functions of defensins, activation of pathwaysresulting in defensin expression and secretion may allow for theantibiotic-independent clearance of bacterial infection, and thus avoidthe generation of bacterial resistance.

In certain embodiments, the compositions described herein may be used totreat and/or prevent one or more bacterial infections of the skin, ordiseases of the skin associated with a bacterial infection. In someembodiments, the compositions described herein are used to treatlocalized skin infections and/or diffuse skin infections. In someembodiments, the compositions described herein are used to treat orprevent skin infections or diseases of the skin associated withbacterial infections affecting the epidermis, dermis, and/orsubcutaneous (hypodermis) tissues of the skin. In some of theseembodiments, the affected layers of the skin include one or more layersof the epidermis (i.e., stratum basale, stratum spinosum, stratumgranulosum, stratum licidum, and stratum corneum), one or more types oftissues of the dermis (i.e., collagen, elastic tissue, and reticularfibers), one or more layers of the dermis (i.e., the upper, papillarylayer and the lower reticular layer); and/or one or more types of tissueof the hypodermis (i.e., fat, elastin and connective tissue). In anembodiment, the compositions described herein are used to treat orprevent bacterial infections on the skin surface. In another embodiment,the compositions described herein are used to treat or prevent aStaphylococcus (“Staph”) infection of the skin and/or a Streptococcus(“Strep”) infection of the skin. In yet another embodiment, thecompositions described herein are used to treat Staphylococcus albusand/or Staphylococcus aureus infection of the skin. In some embodiments,the compositions described herein are used to treat or preventcellulitis, impetigo, folliculitis, erythrasma, carbuncles, furuncles,abscesses, erysipelas, and/or a cutaneous anthrax. In anotherembodiment, the compositions described herein are used to treat orprevent cellulitis. Cellulitis affects the deeper dermis andsubcutaneous tissues, and usually affects the face, arms, and legs, andalmost always occurs due to a break in the skin that leads to abacterial infection. The symptoms of cellulitis include one or more of:swelling of the skin around the break in the skin, pain, tenderness,outward signs of blistering, red lines between the lymph nodes, fever,and chills. In another embodiment, the composition described herein areused to treat or prevent a bacterial infection of the skin at the hairfollicles (e.g., folliculitis). The symptoms of folliculitis includeswelling, pustules surrounding the hair, hard nodules, and pain. Inanother embodiment, the compositions described herein may be used totreat or prevent acne. Appearance of acne is frequently a symptom of abacterial infection. In one embodiment, the compositions describedherein are used to treat or prevent dermatitis associated with or causedby a bacterial infection. In some embodiments, the compositionsdescribed herein prevent the onset or development of one or more of theabove-listed symptoms, or reduce duration and/or severity of one or moreof these symptoms.

In certain embodiments, the compositions described herein may be used totreat and/or prevent one or more intestinal/digestive bacterialinfections or gastrointestinal diseases associated with bacterialinfections. The common forms of intestinal bacterial infection includesalmonella, shigella, E. coli, Clostridium, Staphylococcus, Listeria,and Yersinia. These bacteria cause diarrhea and inflammation of thestomach and intestines, also known as gastroenteritis. Symptoms of anintestinal bacterial infection include but are not limited to abdominalcramps and pain, bloody feces, loss of appetite, nausea sometimesaccompanied by vomiting, fever, and diarrhea. In some embodiments, thecompositions described herein are used to treat a patient displaying oneor more symptoms of food poisoning, which is often associated with abacterial infection.

In certain embodiments, the compositions described herein may be used totreat a disease associated with a Staph infection (e.g., aStaphylococcus aureus infection). In some of these embodiments, thedisease is a Staph infection of the skin, nose, mouth, and/or genitalarea. In some embodiments, the disease is pneumonia, meningitis,endocarditis, toxic shock syndrome, and/or septicemia. In an embodiment,the compositions described herein may be used to treat Staph bacteriaresistant to one or more antibiotics, for example, methicillin resistantStaph aureus (“MRSA”). In certain embodiments, the compositionsdescribed herein are administered to a subject diagnosed with a Staphinfection, or to a subject displaying one or more symptoms of a Staphinfection (e.g., presence of one or more of: small red bumps, crusty redbumps, pus filled bumps or abscess, boils, styes in the eyes, blistersand/or red scabby skin, such as red scabby skin around the nose andmouth, or symptom/s of a Toxic Shock Syndrome). In some embodiments, thecompositions described herein prevent the onset or development of one ormore of the above-listed symptoms, or reduce duration and/or severity ofone or more of these symptoms.

In certain embodiments, the compositions described herein may be used totreat or prevent a cold associated with a bacterial infection. Forexample, the compositions described herein may be used to treat orprevent a cold that persists despite the use of standard pain reliefmedications. In one embodiment, the compositions described herein areused to treat or prevent a bacterial infection of sinus, ear or throat.The symptoms of such bacterial infections include localized pain andswelling. Bacterial infection in the sinus may lead to nasal dischargeand acute pain in parts of the face or forehead. In one embodiment, thecompositions described herein are used to treat strep throat(Streptococcus pyogenes). In some embodiments, the compositionsdescribed herein prevent the onset or development of one or more of theabove-listed symptoms, or reduce duration and/or severity of one or moreof these symptoms.

In some embodiments, the compositions described herein may be used totreat or prevent a genital, urinary tract or anal bacterial infection,or a disease of urinary or reproductive tract associated with abacterial infection. In some of these embodiments, the compositionsdescribed herein may be used to treat a sexually transmitted diseaseassociated with a bacterial infection. Symptoms of such infectionsinclude but are not limited to painful urination, cloudy discharge,and/or pain during intercourse. In some of these embodiments, thecompositions described herein are used to treat or prevent one or moreof syphilis, gonorrhea, chlamydia, and trichomonaisis. In oneembodiment, the compositions described herein are used to treatChlamydia. In another embodiment, the described herein are used to treatgonorrhea. Symptoms of gonorrhea include localized pelvic pain, itchingand irritation, painful urination, a thick yellow or green discharge,bleeding between menstrual periods. In one embodiment, the compositionsdescribed herein are used to treat or prevent a bacterial vaginosisinfection. Symptoms of bacterial vaginosis include vaginal discharge,odor, vaginal itching, and abdominal pain. In some embodiments, thecompositions described herein prevent the onset or development of one ormore of the above-listed symptoms, or reduce duration and/or severity ofone or more of these symptoms.

In other embodiments, the compositions described herein may be used totreat or prevent a respiratory tract infection (e.g., a bacterialinfection of the lungs), or a respiratory disease associated with abacterial infection. In some embodiments, such compositions are used totreat or prevent upper respiratory tract infections. In one embodiment,such compositions are used to treat or prevent tuberculosis.Tuberculosis is caused by mucobacterium tuberculosis, and it is a highlyinfectious disease that is spread from person to person by sneezing orsaliva. Thus, the compositions described herein may be used to treat notonly subjects diagnosed with tuberculosis or displaying symptoms oftuberculosis, but also individuals in contact with such subjects (e.g,family members, caretakers or medical personnel). Symptoms oftuberculosis include coughing blood, excessive weight loss, fatigue,loss of appetite and persistent fever. In some embodiments, thecompositions described herein prevent the onset or development of one ormore of the above-listed symptoms, or reduce duration and/or severity ofone or more of these symptoms. In one embodiment, the compositionsdescribed herein are used to treat pneumonia and/or a Streptococcuspneumoniae infection. In another embodiment, such compositions are usedto treat bronchitis. In one embodiment, the compositions describedherein are used to treat Moraxella catarrhalis, Streptococcus pneumoniaand/or Haemophilus influenza.

In some embodiments, the compositions described herein are used to treator prevent bacterial infections of a mucosal surface (e.g., oralmucosa), or a disease/condition of mucosal surface associated with abacterial infection. In one embodiment, the compositions describedherein are used to treat or prevent bacterial infections of the oralcavity. For example, such compositions may be used to treat or preventconditions associated with bacterial infections in the mouth such asgingivitis, caries, and/or tooth decay. In one embodiment, thecompositions described herein may be used in oral hygiene products.

In one embodiment, the compositions described herein are used to treat abacterial infection of the ear, such as middle ear infection, or adisease associated with such infection. In one embodiment, a compositiondescribed herein is used to treat otitis media caused by a bacterialinfection.

In some embodiments, the compositions described herein are used to treator prevent bacterial infections of implanted prosthesis, such as heartsvalves and catheters. In some embodiments, the compositions describedherein are used to treat animal bites, for example cat or dog bite, inorder to prevent a bacterial infection.

The compositions described herein may be used to treat or prevent avariety of diseases associated with a bacterial infection including butnot limited to leprosy (Hansen's disease), cholera, anthrax (e.g.,cutaneous anthrax, pulmonary anthrax, gastrointestinal anthrax),pertussis, granuloma inguinale, bacterial vaginosis, gonorrhea,ophthalmia neonatorum, septic arthritis, syphilis, congenital syphilis,whooping cough, mycobacterium avium complex, meliodosis, leptospirosis,tetanus, scarlet fever, strep infections, invasive group A Streptococcaldisease, Streptococcal Toxic shock syndrome, meningococcal disease,bacterimia, strep throat, Typhoid fever type salmonellosis, dysentery,colitis, salmonellosis with gastroenteritis and enterocolitis, bacillarydysentery, amebic dysentery, shigellosis, diphtheria, cutaneousdiphtheria, respiratory diphtheria, Legionnaires' disease, tuberculosis,latent tuberculosis, hemophilus influenzae B, typhoid fever, vibrioparahaemolyticus, vibrio vulnificus, vibrio, yersiniosis, Whipple'sdisease, acute appendicitis, meningitis, encephalitis, impetigo,cellulitis, carbuncle, boil, acne, sepsis, septicemia, pneumonia,mycoplasma pneumonia, meningococcal disease, meningitis,Waterhouse-Friderichsen syndrome, ptomaine food poisoning, Staph foodpoisoning, Toxic shock syndrome, necrotizing pneumonia, septicemia,acute infective endocarditis, an infection of sweat glands (e.g.,Hidradenitis suppurativa), a bacterial disease transmitted by a tick(e.g., Rocky Mountain Spotted Fever. Lyme disease), botulism, plague(e.g., bubonic plague, pneumonic plague), tularemia, brucellosis, acuteenteritis, nongonococcal urethritis, lymphogranuloma venerium, trachoma,inclusion conjunctivitis of the newborn, psittacosis, pseudomembranouscolitis, gas gangrene, acute food poisoning, diarrhea, traveller'sdiarrhea, diarrhea in infants, hemorrhagic colitis, hemolytic-uremicsyndrome, bronchitis, listeriosis, anaerobic cellulitis, peptic ulcer,Pontiac fever, cystitis, endometritis, otitis media, sinusitis,streptococcal pharyngitis, rheumatic fever, erysipelas, puerperal fever,necrotizing fasciitis, nosocomial infections, pseudomonas infection,and/or cat scratch disease.

5.5 Patient Populations

In certain embodiments, a sNAG nanofiber composition described hereinmay be administered to a naïve subject, i.e., a subject that does nothave a bacterial infection. In one embodiment, a composition describedherein is administered to a naïve subject that is at risk of acquiring abacterial infection.

In one embodiment, a sNAG nanofiber composition described herein may beadministered to a patient who has been diagnosed with a bacterialinfection. In another embodiment, a composition described herein may beadministered to a patient who displays one or more symptoms of abacterial infection.

In certain embodiments, the compositions described herein areadministered to patients diagnosed with a bacterial infection. Incertain embodiments, a patient is diagnosed with a bacterial infectionprior to administration of a composition described herein. For example,the compositions described herein may be administered to a patient whena bacterial antigen is detected in a biological sample taken from thepatient. In one embodiment, a biological sample is obtained from thesite or area to be treated by the compositions described herein or anarea to which the compositions described herein are to be administered.In one embodiment, a swab is used to collect cells or pus from the siteof the suspected infection to detect a bacterial infection. In anotherembodiment, a fluid is aspirated from the suspected site of an infection(e.g., a wound) to detect a bacterial infection. In yet anotherembodiment, a tissue biopsy is performed to detect a bacterialinfection. In an embodiment where the suspected site of an infection isa wound, a wound culture may be performed to detect a bacterialinfection. In another embodiment, the biological sample is obtained fromblood, urine, sputum or feces of the patient. In some embodiments, ablood or a urine test may be performed to detect a bacterial infection(e.g., when a bacterial infection is suspected to have spread into theblood or other tissues/organs). In some embodiments, the collectedsample (e.g., cells, tissues or fluid) is tested using DNA detectionmethods such as PCR for presence of one or more types of bacteria. Inother embodiments, immunofluorescence analysis, serology, culture (e.g.,blood agar culture), or any other test known and/or practiced in the artmay be used for laboratory diagnosis of bacterial infection.

In other specific embodiments, the compositions described herein may beadministered to a patient diagnosed with or displaying one or moresymptoms of a disease associated with a bacterial infection. In certainembodiments, a patient is diagnosed with a disease associated with abacterial infection or displays one or more symptoms of a diseaseassociated with a bacterial infection prior to administration of acomposition described herein. A disease associated with a bacterialinfection may be diagnosed by any method known to a skilled artisan,including evaluation of the patient's symptoms and/or detection of abacterial antigen in a biological sample of the patient (e.g., asdescribed above). In one example, the compositions described herein maybe administered to a patient diagnosed with a disease associated with abacterial infection by a treating physician or another medicalprofessional. In another example, a patient may use the compositionsdescribed herein upon detection of one or more symptoms of a diseaseassociated with a bacterial infection.

In certain embodiments, a composition described herein is administeredto a patient who has been diagnosed with an infection, e.g., thebacterial infection by Bacillus anthracis, Bordetella pertussis,Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucellamelitensis, Brucella suis, Campylobacter jejuni, Chlamydia psittaci,Chlamydia pneumonia, Chlamydia trachomatis, Clamidophila psittaci,Clostridium botulinum, Clostridium dificule, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Francisella tularensis,Haemophilus influenzae, Helicobacter pylori, Legionella pneumphila,Leptospira pneumophila, Leptospira interrogans, Listeria monocytogenes,Moraxella catarrhalis, Mycobacterium leprae, Mycobacterium tuberculosis,Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides,Pseudomonas aeruginosa, Proteus mirabilis, Pneumocystis jiroveci,Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcuspneumonia, Streptococcus pyogenes, Treponema pallidum, Vibria cholerae,Yersinia pestis and/or any other bacterial infection described herein orknown in the art. In one embodiment, a composition described herein isadministered to a patient who has been diagnosed with the bacterialinfection by MRSA or Pseudomonas aeruginosa.

In certain embodiments, a composition described herein is administeredto a patient who has been diagnosed with a disease associated withbacterial infection, e.g., leprosy (Hansen's disease), cholera, anthrax(e.g., cutaneous anthrax, pulmonary anthrax, gastrointestinal anthrax),pertussis, granuloma inguinale, bacterial vaginosis, gonorrhea,ophthalmia neonatorum, septic arthritis, syphilis, congenital syphilis,whooping cough, mycobacterium avium complex, meliodosis, leptospirosis,tetanus, scarlet fever, strep infections, invasive group A Streptococcaldisease, Streptococcal Toxic shock syndrome, meningococcal disease,bacterimia, strep throat, Typhoid fever type salmonellosis, dysentery,colitis, salmonellosis with gastroenteritis and enterocolitis, bacillarydysentery, amebic dysentery, shigellosis, diphtheria, cutaneousdiphtheria, respiratory diphtheria, Legionnaires' disease, tuberculosis,latent tuberculosis, hemophilus influenzae B, typhoid fever, vibrioparahaemolyticus, vibrio vulnificus, vibrio, yersiniosis, Whipple'sdisease, acute appendicitis, meningitis, encephalitis, impetigo,cellulitis, carbuncle, boil, acne, sepsis, septicemia, pneumonia,mycoplasma pneumonia, meningococcal disease, meningitis,Waterhouse-Friderichsen syndrome, ptomaine food poisoning, Staph foodpoisoning, Toxic shock syndrome, necrotizing pneumonia, septicemia,acute infective endocarditis, an infection of sweat glands (e.g.,Hidradenitis suppurativa), a bacterial disease transmitted by a tick(e.g., Rocky Mountain Spotted Fever, Lyme disease), botulism, plague(e.g., bubonic plague, pneumonic plague), tularemia, brucellosis, acuteenteritis, nongonococcal urethritis, lymphogranuloma venerium, trachoma,inclusion conjunctivitis of the newborn, psittacosis, pseudomembranouscolitis, gas gangrene, acute food poisoning, diarrhea, traveller'sdiarrhea, diarrhea in infants, hemorrhagic colitis, hemolytic-uremicsyndrome, bronchitis, listeriosis, anaerobic cellulitis, peptic ulcer,Pontiac fever, cystitis, endometritis, otitis media, sinusitis,streptococcal pharyngitis, rheumatic fever, erysipelas, puerperal fever,necrotizing fasciitis, nosocomial infections, pseudomonas infection,and/or cat scratch disease.

In some embodiments, a composition described herein is administered to apatient with a bacterial infection before symptoms of the infectionmanifest or before symptoms of the infection become severe (e.g., beforethe patient requires treatment or hospitalization). In some embodiments,a composition described herein is administered to a patient with adisease after symptoms of the disease manifest or after symptoms of thedisease become severe (e.g., after the patient requires treatment orhospitalization).

In some embodiments, a subject to be administered a compositiondescribed herein is an animal. In certain embodiments, the animal is abird. In certain embodiments, the animal is a canine. In certainembodiments, the animal is a feline. In certain embodiments, the animalis a horse. In certain embodiments, the animal is a cow. In certainembodiments, the animal is a mammal, e.g., a horse, swine, mouse, orprimate, preferably a human. In some embodiments, the animal is a pet ora farm animal.

In certain embodiments, a subject to be administered a compositiondescribed herein is a human adult. In certain embodiments, a subject tobe administered a composition described herein is a human adult morethan 50 years old. In certain embodiments, a subject to be administereda composition described herein is an elderly human subject.

In certain embodiments, a subject to be administered a compositiondescribed herein is a premature human infant. In certain embodiments, asubject to be administered a composition described herein is a humantoddler. In certain embodiments, a subject to be administered acomposition described herein is a human child. In certain embodiments, asubject to be administered a composition described herein is a humaninfant. In certain embodiments, a subject to whom a compositiondescribed herein is administered is not an infant of less than 6 monthsold. In a specific embodiment, a subject to be administered describedherein is 2 years old or younger.

In yet other embodiments, a composition described herein may beadministered to a patient who is at risk (e.g., at high risk) ofdeveloping a bacterial infection. Patients that are at high risk ofdeveloping a bacterial infection include but are not limited to theelderly (e.g., human elderly) and immunocompromised. In someembodiments, a composition described herein is administered to a patientat risk of developing a bacterial infection, such as but not limited toan immunosuppressed patient, (e.g., as a result of cancer treatment or atransplantation procedure), a human child, a premature human infant, anelderly human, a person with diabetes, a person diagnosed with cancer, apatient who has been treated with a course of traditional antibiotics, apatient who has undergone a surgery, and or a patient with a wound. Insome embodiments, the compositions described herein may be administeredprophylactically to patients who are at risk for developing a bacterialinfection, e.g., those with compromised immune systems due to, forexample, age, malnourishment, disease, chemotherapy, those who have beentreated with a course of traditional antibiotics, or those who have awound (e.g., an open wound). In other embodiments, a patient at risk ofdeveloping a bacterial infection is an HIV/AIDS patient, a cancerpatient, a patient who has undergone a transplant procedure, a patientwith asthma (e.g., severe asthma), a drug user, a patient with adermatologic conditions, a patients with an invasive device (e.g., anintravascular catheter), a health care workers or a patient who spendstime in a confined facility (e.g., a prison inmate, a soldier, a patientin a long-term healthcare facility such as a nursing home, etc.). Apatient to be administered a composition described herein may also be apatient with a chronic obstructive pulmonary disorder (COPD), emphysema,rhinitis, bronchitis, laryngitis, tonsillitis, and/or cystic fibrosis.

In certain embodiments, a composition described herein is administeredto a patient who has not been diagnosed with a viral infection (e.g.,HIV/AIDS), a fungal infection, or an yeast infection. In certainembodiments, a composition described herein is administered to a patientwho does not belong to one or more of the following patient groups: animmunocompromised patient, a cancer patient, an HIV/AIDS patient, apatient with asthma, a patient who has undergone a transplant procedure,a patient who has undergone a surgery, and or a patient with a wound. Inone embodiment, the patient to be administered a composition describedherein does not have a wound (e.g., a chronic wound, or an open wounddue, e.g., to a surgery or battlefield trauma).

In certain embodiments, a subject to be administered a compositiondescribed herein is a subject with no or low level of expression of oneor more defensin peptides or a mutation/deletion in a gene or genesencoding one or more defensin peptides. In some embodiments, a subjectto be administered a composition described herein is a subject with noor low or altered level of expression of one or more α-defensins (e.g.,DEFA1, DEFA1B, DEFA3, DEFA4, DEFA5, DEFA6), one or more β-defensins(e.g., DEFB1, DEFB2, DEFB4, DEFB103A, DEFB104A, DEFB105B, DEFB107B,DEFB108B, DEFB110, DEFB112, DEFB114, DEFB118, DEFB119, DEFB123, DEFB124,DEFB125, DEFB126, DEFB127, DEFB128, DEFB129, DEFB131, DEFB136), and/orone or more β-defensins (e.g., DEFT1P). In some embodiment, a subject tobe administered a composition described herein is a subject with no orlow or altered level of expression of one or more of DEFA1, DEFA3,DEFA4, DEFA5, DEFB1, DEFB3, DEFB103A. DEFB104A, DEFB08B, DEFB112,DEFB114, DEFB118, DEFB119, DEFB123, DEFB124, DEFB125, DEFB126, DEFB128,DEFB129 and DEFB131. In certain embodiments, a subject to beadministered a composition described herein is a subject with no or lowor altered level of expression of one or more Toll receptors (e.g.,TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,and/or TLR12). In yet other embodiments, a subject to be administered acomposition described herein is a subject with no or low or alteredlevel of expression of one or more of IL-1, CEACAM3, SPAG11, SIGIRR(IL1-like receptor), IRAK1, IRAK2, IRAK4, TBK1, TRAF6 and IKKi. In someembodiments, a subject to be administered a composition described hereinis a subject with no or low or altered level of expression of one ormore of IRAK2, SIGIRR. TLR1, TLR2, TLR4, TLR7, TLR8, TLR10 and TRAF6. Alow level of expression of a gene is a level that is lower (e.g., morethan 1.25 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold lower) thanthe normal level of expression, wherein the normal level of expressionis the level of expression is considered normal in the species to whichthe subject belongs by a skilled artisan and/or the level of expressionin the majority of the subjects of the same species. An altered level ofexpression of a gene is a level that differs (e.g., by more than 20%,25%, 30%, 50%, 75%, 100%, 150%, 200%, 250%, 300%) from the normal levelof expression, wherein the normal level of expression is the level ofexpression is considered normal in the species to which the subjectbelongs by a skilled artisan and/or the level of expression in themajority of the subjects of the same species. Wherein the “normal”expression of one or more defensin genes is: (i) the average expressionlevel known to be found in subjects not displaying symptoms or notdiagnosed with the disease or infection to be treated; (ii) the averageexpression level detected in three, five, ten, twenty, twenty-five,fifty or more subjects not displaying symptoms or not diagnosed with thedisease or infection to be treated; and/or (iii) the level of expressiondetected in a patient to be administered a composition described hereinbefore the onset of the disease or infection.

5.6 Modes of Administration of sNAG Nanofiber Compositions

In certain embodiments, methods are described herein for treating orpreventing a bacterial infection or a disease associated with abacterial infection, wherein a composition comprising the sNAGnanofibers is topically administered to a patient in need of suchtreatment. In some embodiments, a sNAG nanofiber composition is appliedtopically to tissue or organ which has an increased risk of a bacterialinfection or disease.

In some embodiments, an effective amount of the sNAG nanofibers and/or asNAG nanofiber composition is administered to a subject.

In some embodiments, a composition comprising the sNAG nanofibers isadministered topically to the site of the bacterial infection in apatient or to the site affected by a disease associated with bacterialinfection. In yet other embodiments, a composition comprising the sNAGnanofibers is administered topically to the site and around the site ofthe bacterial infection in a patient or to the site affected by adisease associated with bacterial infection. In yet other embodiments, acomposition comprising sNAG nanofibers is applied in proximity to thesite of the bacterial infection in a patient or in proximity to the siteaffected by a disease associated with bacterial infection. In yetanother embodiment, a composition comprising the sNAG nanofibers isadministered topically to the site at high risk of a bacterialinfection.

The sNAG nanofiber compositions described herein may be administered byany of the many suitable means of topical administration which are wellknown to those skilled in the art, including but not limited totopically to the skin, topically to any other surface of the body (e.g.,mucosal surface), by inhalation, intranasally, vaginally, rectally,buccally, or sublingually. The mode of topical administration may varydepending upon the disease to be treated or prevented. The sNAGnanofiber compositions can be formulated for the various types oftopical administration.

In one embodiment, a composition comprising sNAG nanofibers is appliedto the skin of a patient. For example, such composition may be appliedtopically to the skin of a patient for treating and/or preventing abacterial infection of the skin or a disease of the skin that isassociated with a bacterial infection.

In another embodiment, a composition described herein may be appliedtopically to a mucosal surface of a patient. For example, suchcomposition may be applied topically to oral mucosa for treating and/orpreventing a bacterial infection of the mouth or gums or a disease ofthe mouth or gums that is associated with a bacterial infection.

In some embodiments, a composition comprising sNAG nanofibers is appliedto the wound in a patient. For example, such composition may be appliedtopically directly to site of the wound or in proximity to the site ofthe wound of a patient for treating and/or preventing a bacterialinfection of the wound or a disease associated with a bacterialinfection of the wound. In one such embodiment, the wound is abacterially infected wounds, for example, as diagnosed by one of themethods described herein. The wound may be any one of the types ofwounds described herein. In yet other embodiments, a compositioncomprising sNAG nanofibers is not applied to a wound in a patient, or isnot applied to a bacterially infected wound in a patient.

In some embodiments, a composition described herein may be appliedtopically to a genital, urinal or anal surface/area of a patient. Forexample, such composition may be applied topically to genital, urinal oranal surface/area for treating and/or preventing genital, urinal or analbacterial infections or a disease of such tissues that is associatedwith a bacterial infection.

The above-listed methods for topical administration may includeadministration of the sNAG nanofiber in the form of a cream, anointment, a gel, a liquid solution, a membrane, a film, a spray, apaste, a powder or any other formulation described herein or known inthe art. The sNAG nanofiber may also be applied in a dressing or abandage, for example to treat localized infections/conditions on theskin of a patient.

In some embodiments, a composition described herein may be applied as aspray into the oral cavity and/or respiratory system of a patient. Forexample, such composition may be applied as a spray for treating and/orpreventing bacterial infection of the mouth, nose, gums, throat or lungsor a disease/condition of the mouth, nose, gums, throat or lungs that isassociated with a bacterial infection. In one such embodiment, thecomposition may be formulated to be administered as an inhaler.

In some embodiments, a composition described herein may be applied as asuppository in the rectum, vagina or urethra of a patient. For example,such composition may be applied as a suppository for treating and/orpreventing bacterial infection of the digestive tract, urinary tract orreproductive tract or a disease of such tissues that is associated witha bacterial infection.

In another embodiment, a composition described herein may be applied atthe site of a surgical procedure. For example, such composition may besprayed, applied as a cream, ointment, gel, membrane, or powder, orcoated on the surface of the tissue or organ to be subjected to asurgical procedure or that has been subjected to the surgical procedure.In one embodiment, a composition described herein is applied at the siteof the surgical incision, at the site of the excised tissue, or at thesite of surgical stitches or sutures. Such administration of acomposition described herein may prevent a post-surgical infection. Forexample, a composition described herein may be used during or after asurgical procedure which is known to pose high risk of a bacterialinfection. Surgical procedures that are known to pose high risk of abacterial infection include bowel resection, gastrointestinal surgicalprocedures, kidney surgery, etc. A composition described herein may beapplied at the site of any of the above-listed or other surgicalprocedures.

In yet other embodiments, a composition described herein may be coatedon a device, for example an oral hygiene product, a catheter, a surgicalinstrument or another product, to be used in or inserted into a patient,in order to prevent a bacterial infection in a patient.

In some embodiments, methods contemplated herein include a step thatincludes detection/diagnosis of a bacterial infection in a patient. Insome embodiments, detection/diagnosis involves a test or assay for oneor more bacteria or bacterial antigens in a biological sample of thepatient. In other embodiments, diagnosis involves assessing whether thepatient has one or more symptoms of a bacterial infection or a diseaseassociated with a bacterial infection.

The compositions described herein may exhibit sustained releaseproperties and/or may be administered in a formulation resulting in asustained release of such compositions. In some embodiments, the sNAGnanofibers biodegrade over time as described in Section 5.1, supra, andthese properties of sNAG nanofibers may lead to or contribute tosustained release of the compositions described herein. In yet otherembodiments, the compositions described herein are formulated to displaysustained release capabilities using any methods known in the art. Thecompositions described herein may exhibit sustained release over a timeperiod equal to or more than about 6 hours, 12 hours, 18 hours, 24 hours(1 day), 2 days, 3 days, 5 days, 7 days (1 week), 10 days, 14 days (2weeks), 3 weeks or 4 weeks after administration of the composition tothe patient.

Contemplated treatment regimes include a single dose or a singleapplication of a sNAG nanofiber composition (e.g., of a cream, amembrane or a dressing), or a regiment of multiple doses or multipleapplications of a sNAG nanofiber composition. A dose or an applicationmay be administered hourly, daily, weekly or monthly. For example, adose of a sNAG nanofiber composition may be administered once a day,twice a day, three times a day, four times a day, five times a day,every 3 hours, every 6 hours, every 12 hours, every 24 hours, every 48hours, every 72 hours, once a week, 2 times a week, 3 times a week,every other day, once in 2 weeks, once in 3 weeks, once in 4 weeks, oronce a month.

A sNAG nanofiber composition may be administered for a duration equal toor greater than 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 2.5years, 3 years, 4 years, 5 years, 7 years, 10 years or more. In one suchembodiment, a sNAG nanofiber composition does not cause any side effectsor causes only mild side effects during the duration of the treatment.In one such embodiment, a sNAG nanofiber composition does not lose itseffectiveness or does not cause generation of resistant strains ofbacteria in response to the treatment. In another embodiment, a sNAGnanofiber composition does not cause irritation (e.g., moderate orsevere irritation) or allergy (e.g., moderate or severe allergy).

Concentration of the sNAG nanofiber in a composition may vary. Ingeneral, an effective amount of the sNAG nanofiber is used. A effectiveamount may be an amount sufficient to achieve one or more of the effectsdescribed herein, for example an amount effective to reduce or eradicatea bacterial infection, or reduce or eradicate one or more symptoms of abacterial infection. For example, a composition may comprise about 0.2to 20 mg/cm² of the sNAG nanofibers per dose/application of thecomposition in a form suitable for topical delivery to a patient. Incertain embodiments, a composition described herein comprises about 0.25to 20 mg/cm², about 0.5 to 20 mg/cm², about 1 to 20 mg/cm², about 1 to15 mg/cm², about 1 to 12 mg/cm², about 1 to 10 mg/cm², about 1 to 8mg/cm², about 1 to 5 mg/cm², about 2 to 8 mg/cm², or about 2 to 6 mg/cm²of the sNAG nanofibers per dose/application of the composition in a formsuitable for topical delivery to a patient.

5.7 Combination Therapy

The sNAG nanofiber compositions may be administered in conjunction withother therapies such as substances that boost the immune system,antibacterial agents (e.g., an antibiotic), defensin peptides,defensin-like peptides, pain relief therapy (e.g., an analgesic), feverrelief therapy, and/or other agents or drugs known to be effectiveagainst or commonly used for treatment and/or prevention of bacterialinfections or diseases associated with bacterial infections.

In some embodiments, a composition described herein is administered inconjunction with an additional anti-bacterial agent, for example anantibiotic. In one such embodiment, a composition described herein maybe used to treat a bacterial infection or a disease associated with abacterial infection in conjunction with a standard therapy commonly usedto treat such bacterial infection or such disease. In one embodiment, acomposition described herein may be administered to a patient diagnosedwith or displaying symptoms of a bacterial infection or a diseaseassociated with a bacterial infection in conjunction with a standardanti-bacterial agent (e.g., an antibiotic) known to be effective againstsuch bacterial infection or such disease.

In certain embodiments, a composition described herein is administeredin conjunction with an antibiotic of one of the following classes ofantibiotics: microlides (e.g., erythromycin, azithromycin),aminoglycosides (e.g., amikacin, gentamicin, neomycin, streptomycin),cephalosporins (e.g., cefadroxil, cefaclor, cefotaxime, cefepime),fluoroquinolones (e.g., ciprofloxacin, levofloxacin), penicillins (e.g.,penicillin, ampicillin, amoxicillin), tetracyclines (e.g., tetracycline,doxycycline), and carbapenems (e.g., meropenem, imipenem). In someembodiments, a composition described herein is administered inconjunction with an agent (e.g., an antibiotic) effective to treat orprevent or commonly used to treat or prevent an S. aures infection, MRSAinfection, a Pseudomonas infection, or a C. dificule infection.

In a specific embodiment, a composition described herein is administeredin conjunction with one or more of vancomycin, sulfa drug (e.g.,co-trimoxazole/trimethoprim-sulfamethoxazole), tetracycline (e.g.,doxycycline, minocycline), clindamycin, oxazolidinones (e.g.,linezolid), daptomycin, teicoplanin, quinupristin/dalfopristin(synercid), tigecycline, allicin, bacitracin, nitrofurantoin, hydrogenperoxide, novobiocin, netilmicin, methylglyoxal, and bee defensin-1. Acomposition described herein may also be administered in conjunctionwith a dressing comprising one or more of hydrogen peroxide, tobramycin,chlorhexidine digluconate, chlorhexidine gluconate, levofloxacin, andsilver. In one embodiment, a composition described herein isadministered with one or more of the listed agents to treat or prevent aS. aureus infection, and particularly, an MRSA infection.

In some embodiments, the compositions described herein are administeredbefore (e.g., 1 minute, 15 minutes, 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 12 hours, 24 hours or more before, or any time period inbetween), simultaneously with, or after (e.g., 1 minute, 15 minutes, 30minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours or moreafter, or any time period in between) administration of another therapy.For a example, such compositions maybe administered before,simultaneously with or after administration of an anti-bacterial agent(e.g., an antibiotic).

In some of these embodiments, the compositions described herein may beadministered to a patient to treat or prevent a bacterial infection or adisease associated with bacterial infection after the patient hasundergone a course of treatment of the bacterial infection with anotheranti-bacterial agent (e.g., an antibiotic). In some embodiments, thecompositions described herein may be administered to a patient who hasdeveloped resistance to one or more anti-bacterial agents (e.g., anantibiotic). In one embodiment, the compositions described herein may beadministered to a patient who has undergone a course of treatment withan antibiotic (e.g., an antibiotic standardly used for the treatment ofsuch bacterial infection) and developed resistance to such antibiotic.

However, in certain embodiments, a sNAG nanofiber composition isadministered alone. In one such embodiment, a sNAG nanofiber compositionis not administered with any other therapies, for example, it is notadministered with an immunomodulator, an antibacterial agent (e.g., anantibiotic), a defensin peptide, a defensin-like peptide, a pain relieftherapy (e.g., an analgesic), or a fever relief therapy. In oneembodiment, a sNAG nanofiber composition is not administered inconjunction with an antibiotic. In certain embodiments, sNAG nanofibercompositions are not administered in conjunction with an anti-viralagent, an anti-fungal agent or an anti-yeast agent.

5.8 Kits

A pharmaceutical pack or kit which comprises any of the above-describedsNAG compositions is also contemplated. The pack or kit may comprise oneor more containers filled with one or more ingredients comprising thecompositions described herein. The composition is preferably containedwithin a sealed, water proof, sterile package which facilitates removalof the composition without contamination. Materials from whichcontainers may be made include aluminum foil, plastic, or anotherconventional material that is easily sterilized. The kit can containmaterial for a single administration or multiple administrations of thecomposition, preferably wherein the material for each administration isprovided in a separate, waterproof, sterile package.

In another embodiment, a container having dual compartments is provided.A first compartment contains any of the above-described sNAGcompositions, while the second compartment contains another active agentsuch as another anti-bacterial agent. In the field or the clinic, thecomposition in the first compartment can be readily combined with theagent in the second compartment for subsequent administration to apatient.

Additionally, a kit designed for emergency or military use can alsocontain disposable pre-sterilized instruments, such as scissors,scalpel, clamp, tourniquet, elastic or inelastic bandages, or the like.

Optionally associated with such kit or pack can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. For example, a kit can comprise a notice regarding FDAapproval and/or instructions for use.

The kits encompassed herein can be used in the above applications andmethods.

6. EXAMPLES 6.1 Example 1 sNAG Nanofibers from a Marine Diatom PromoteWound Healing and Defensin Expression Via an Akt1/Ets1-Dependent Pathway

This example demonstrates that sNAG nanofibers promote cutaneous woundhealing and expression of defensins, and that the Akt1→Ets1 pathwayplays a central role in the regulation of cutaneous wound healing bysNAG nanofibers.

6.1.1 Materials and Methods

sNAG/Taliderm nanofibers are produced and supplied by Marine PolymerTechnologies and formed into suitable patches for wound treatment.Wildtype C57 Black and Akt1 null mice were housed at the MedicalUniversity of South Carolina animal facilities. Wildtype and Akt1 nullmice, ages ranging from eight to 12 weeks, were anesthetized with 50%pure oxygen and 50% isoflurane gas. Immediately before wounding, NairHair Removal Lotion was applied to their dorsum to remove any unwantedhair. A dorsal 4 mm circular area of skin was removed using an excisionbiopsy punch. Taliderm was placed onto each wound at day 0 or woundswere left untreated. At days 1, 3, 5, and 7 the wounds werephotographed, measured, and excised using an 8 mm biopsy punch to ensurecomplete removal of the wound and surrounding skin. Wildtype and Akt1null wounds with and without Taliderm treatment were embedded inparaffin in preparation for H&E and immunofluorescent staining.

Paraffin-embedded sections were sectioned and placed on microscopeslides for staining. Slides were washed with xylenes to remove paraffinand rehydrated through a series of graded alcohols. The sections werethen incubated in 0.1% Triton x100 for permeabilization. Sections wereincubated in a boiling Antigen Retrival solution. 1% Animal serum wasused for blocking before incubating in the primary goat antibody,β-defensin 3 1:400 dilution. The sections were then incubated in theprimary antibody overnight at 4° in a humidity chamber. Animmunofluorescent secondary Donkey α-goat 488 antibody 1:200 dilutionwas used, followed by nuclear staining with TOPRO-3. Images werecaptured using confocal microscopy.

Hematoxylin and eosin staining was used to visualize basic structuressuch as the epidermis, dermis, muscle, and blood vessels and todetermine the orientation and approximate location in the wound. H&Estaining was also used to begin to identify which cell types arestimulated by Taliderm in an Akt1-independent manner.

Other materials and methods are described in figure descriptions and inthe results section below, and performed in accordance with the methodsknown in the art.

6.1.2 Results

sNAG Nanofibers Stimulate Akt1 Activation, an Upstream Regulator ofEts1.

FIG. 1A shows a Western blot analysis of phospho-Akt in response to NAGand sNAG stimulation of serum starved EC. FIG. 1B shows RT-PCR analysisof EC infected either with scrambled control or Akt1 shRNA lentivirusesand assessed for expression of Ets1 and S26 as a loading control. FIG.1C illustrates a signal transduction pathway transducing a signal fromsNAG nanofibers to Akt1, Ets1 and Defensins.

Delayed Wound Healing in Akt1 Null Animals is Partially Rescued byTaliderm (sNAG) Treatment.

FIG. 2A shows representative images of wounded WT and AKT1 null micewith and without treatment of Taliderm. FIG. 2B shows H&E staining ofrepresentative mouse skin sections from day 3 wounds. H&E staining ofwildtype and Akt1 wound excisions indicate a Taliderm dependent increasein keratinocyte proliferation and migration. The dashed lines indicatethe area of keratinocyte proliferation across the wound margin. In boththe wildtype and Akt1 treated wounds there is an evident increase inreepithelization across the wound margin compared to the wildtype andAkt1 control. This indicates that Taliderm increases kertainoctyerecruitment independent of the Akt pathway. Although Taliderm induces acomplete reepithlization of the epidermis across the wound margin, thereis still substantial lack of revascularization in the underlying tissuecompared to the wildtype. This is evident by substantial hemorrhagingand infiltration of red blood cells in the Akt1 aminals.

sNAG Nanofibers Stimulate Cytokine and Defensin Expression in PrimaryEndothelial Cells.

FIG. 3A shows immunohistochemistry of EC treated with or without sNAGusing an antibody directed against a-defensin. FIG. 3B presents ELISAshowing that nanofiber treatment of EC results in the secretion ofα-defensins 1-3.

sNAG Nanofibers Stimulate Defensin Expression in Primary EndothelialCells in an Akt1 Dependent Manner.

FIGS. 4A and 4B show quantitative RT-PCR analyses of serum starved ECtreated with or without sNAG, with or without PD98059 (MAPK inhibitor),Wortmannin (PI3K inhibitor) or infected with a scrambled control or Akt1shRNA lentiviruses and assessed for expression of the genes indicated.

sNAG Nano Fibers Stimulate β-Defensin 3 Expression in MouseKeratinocytes.

FIG. 5A shows immunofluorescent staining with β-defensin 3 andInvolucrin antibodies of paraffin embedded mouse cutaneous woundsections from WT and Akt1 null animals on Day 3. A cutaneous woundhealing model was developed in both WT and Akt1 null mice to assess theeffects of Taliderm in vivo. These findings show that β-defensin 3expression increases in Taliderm treated animals in an Akt1-dependentmanner. The ability of Taliderm to increase defensin expression in ahealing wound has important implications for treating and controllingwound infection. FIG. 5B shows quantification of β-defensin 3immunofluorescent staining using NIHImageJ software. FIG. 5C showsimmunofluorescent staining of WT and Akt1 null treated and untreatedkeratinocytes with β-Defensin 3 and TOPRO-3. Notice the increase ingreen β-Defensin 3 staining in WT and Akt1 Taliderm treated wounds. Theimmunofluorescent labeling of wound sections illustrates that Talidermtreated wounds show an increase in β-defensin 3 expression in an Akt1dependent manner. Although the Akt1 treated wounds show a reasonableincrease in β-defensin 3, the wildtype treated wounds illustrate a moreremarkable increase. This indicates that 1-defensin 3 expression is notonly increased by application of the nanofiber, but is at leastpartially dependent on the Akt1 pathway. β-defensin 3 expression seemslimited to the keratinocytes indicating this expression is keratinocytespecific.

Akt1 Dependent Transcription Factor Binding Sites.

FIG. 6 shows schematic of Akt1 dependent transcription factor bindingsites. Using Genomatix software, 500 bp upstream of the transcriptionstart site was analyzed for conserved sites on the mRNA of DEF1, 4, and5.

6.1.3 Conclusions

The provided data show that sNAG nanofiber stimulation of Ets1 resultsfrom the activation of Akt1 by these nanofibers. Nanofiber treatmentresulted in marked increases in the expression of genes involved incellular recruitment, such as IL-1 (a known Ets1 target), VEGF andseveral defensins (β3, α1, α4, and α5), small anti-microbial peptidesrecently shown to act as chemoattractants. Both pharmacologicalinhibition of the PI3K/Akt1 pathway and Akt1 knockdown using shRNAsresulted in decreased expression of these chemotactic factors. Akt1 nullmice exhibited a delayed wound healing phenotype that is partiallyrescued by Taliderm nanofibers. Taliderm treated wounds also showed anincrease in defensin expression that is Akt1 dependent.

The increase of β-defensin 3 expression and keratinocyte proliferationin Taliderm treated wounds demonstrates the beneficial use of Talidermas an effective wound healing product. Taliderm acts to increaseanti-microbial peptide expression in keratinocytes in an Akt1 dependentmanner suggesting the essential role of Akt1 in the function of sNAGnanofibers. This correlates with the results from other studies in thelaboratory (Buff, Muise-Helmericks, unpublished) that inhibition of thePI3K/Akt1 pathway and Akt1 knockdown using shRNAs results in decreasedexpression of these chemotactic factors.

Although the increased expression of tβ-defensin 3 is Akt1-dependent,H&E staining of 8 mm wound excisions (FIG. 2B) indicated that Talidermacts independent of Akt1 in wound reepithelization. Even though the newkeratinocytes span the entire wound margin, the underlying tissue didnot demonstrate the same stimulation in vascular growth. This indicatesthe that absence of Akt1 is responsible for leaky blood vessels and thelarge amount of floating red blood cells in the dermis. This suggeststhat Taliderm is dependent on the Akt1 pathway for an increase invascularization.

In summary, (i) sNAG nanofibers (Taliderm) increase wound healing inpart by stimulating angiogenesis; (ii) sNAG nanofibers treatment ofendothelial cells activate an Akt1/Ets1 dependent pathway leading tochanges in cell motility and cytokine secretion; (iii) Taliderm treatedwounds show increased expression of β-defensin 3 in an AKT1 dependentmanner; (iv) treatment of Akt1 null animals with Taliderm partiallyrescues the phenotype, leading to markedly increased keratinocyteproliferation/migration; and (v) bioinformatics analysis indicates thatETS1 is likely involved in the sNAG activated pathway leading toincreased wound healing and cytokine secretion.

Taken together these findings suggest a central role of the Akt1-Ets1pathway in the regulation of cutaneous wound healing by sNAG nanofibersand support the use of these nanofibers as a novel and effective methodfor enhancing wound healing.

6.2 Example 2 sNAG Nanofibers Increase Defensin Expression, IncreaseKinetics of Wound Closure, and have an Indirect Defensin-DependentAnti-Bacterial Effect

This example demonstrates that sNAG nanofibers have a potentanti-bacterial effect against Staphylococcus aureus in vivo, which isindirect and defensin-dependent. This example also shows that sNAGnanofibers induce expression of defensins in vitro in keratinocytes andendothelial cells and in vivo in cutaneous wounds, in an Akt-1 dependentmanner, and increase the kinetics of wound closure.

6.2.1 Materials and Methods

Tissue Culture, Pharmacological Inhibition, ELISA:

Human umbilical cord vein EC (Lonza) were maintained at 37° with 5% CO2in endothelial basal medium 2 (Lonza). Endothelial basal medium 2 (EBM2)was supplemented with EC growth medium 2 SingleQuots as described byLonza procedures and 1% penicillin/streptomycin (Invitrogen). Serumstarvation was performed at 80-90% confluency in EBM2 supplemented with0.1% fetal calf serum (Valley Biomedical) for 24 hours followed bystimulation with highly purified pGlcNAc (50 μg/ml) nanofibers (sNAG) insterile water (provided by Marine Polymer Technologies, Inc., Danvers,Mass., USA). The pGlcNAc diatom-derived nanofibers used in this studyare short biodegradable fibers derived from a longer form (NAG), andhave an average length of 4-7 μm and a polymer molecular weight ofapproximately 60,000 Da. For inhibition using PD098059 (50 μM) orwortmannin (100 nM), cells were pre-treated for 45 minutes prior to 3hour stimulation with sNAG (50 μg/ml).

Statistical Analysis:

Each quantitative experiment was performed at least in triplicate atleast three independent times. All statistical analyses were performedusing Microsoft Excel to calculate means, standard deviations andstudent t-test

Lentiviral Infection:

Mission shRNA lentiviral constructs directed against Akt1 were purchasedfrom Sigma/Aldrich. A scrambled pLKO.1 shRNA vector was purchased fromAddgene. Lentiviruses were propagated in 293T cells, maintained in DMEMsupplemented as above. Lentiviral production was performed using psPAX2and pMD2.G packaging vectors purchased from Addgene using the protocolfor producing lentiviral particles from Addgene. For infection of targetcells, 7.5×10⁵ cells were plated on 100 mm2 plates and allowed toincubate overnight. The next day, cells were transduced using a finalconcentration of 1 μg/ml polybrene and either scrambled control or Akt1shRNA lentiviruses. After transduction, endothelial cells were serumstarved overnight and stimulated with sNAG (50 g/ml) for 3 hours. Allinfections were monitored for appropriate knockdown by RT-PCR.

RT-PCR:

For semi-quantitative RT-PCR, RNA was extracted with RNAsol (Teltest,Inc.) following manufacturer's instructions. cDNA was synthesized from 2μg total RNA with a Superscript First Strand Synthesis Kit (Invitrogen),using Oligo(dT) following the manufacturer's instructions. PCR reactionscontained equal amounts of cDNA and 1.25 μM of the appropriate primerpair (Sigma-Proligo, St. Louis, Mo., USA). All primer sequences used inthese analyses are as follows:

(SEQ ID NO: 1) Akt1 F 5′ GAGGCCGTCAGCCACAGTCTC 3′ (SEQ ID NO: 2) Akt1 R5′ ATGAGCGACGTGGCTATTGTG 3′ (SEQ ID NO: 3) β-Defensin3 F 5′GTGGGGTGAAGCCTAGCAG 3′ (SEQ ID NO: 4) β-Defensin3 R 5′TTTCTTTCTTCGGCAGCATT 3′ (SEQ ID NO: 5) α-Defensin1 F 5′CACTCCAGGCAAGAGCTGAT 3′ (SEQ ID NO: 6) α-Defensin1 R 5′TCCCTGGTAGATGCAGGTTC 3′ (SEQ ID NO: 7) S26 F 5′ CTCCGGTCCGTGCCTCCAAG 3′(SEQ ID NO: 8) S26 R 5′ CAGAGAATAGCCTGTCTTCAG 3′

Cycling conditions were: 94° C. for 5 min; 30-35 cycles of 94° C. for 1min, 55-65° C. (based on primer T_(m)) for 1 min, 72° C. for 1 min; 72°C. for 7 min and cooled to 4° C. Cycle number was empirically determinedto be within the linear range of the assay for each primer pair used.All semi-quantitative RT-PCR was performed with the ribosomal proteinsubunit S26 primers as internal controls. Products were visualized on aBioRad Molecular Imaging System (Hercules, Calif., USA). Real time PCRwas performed using a Brilliant CYBR green QPCR kit in combination withan Mx3000P Real-Time PCR system both purchased from Stratagene. Primersdetecting the ribosomal subunit S26 were used as internal controls.

Excisional Wound Healing Model:

Wild Type C57Bl/6 and Akt1−/− [43] were used in all experiments. TheAkt1 null animals were created using an insertional mutagenesis strategyat the translational start site that blocks expression of the entireprotein. Wounding was performed on anesthetized adult male mice between8-12 weeks old. Two full thickness cutaneous wounds were created using a4 mm biopsy punch (Miltex), to create two identical wounds on eachflank. Mice were anesthetized using an O₂/Isoflurane vaporizinganesthesia machine (VetEquip, Inc.). Isoflurane was used at 4% forinduction; 2% for surgery. Prior to surgery hair was removed bydepilation and the area was washed and sterilized using 70% ethanol.Wounds were either treated with sNAG membrane moistened with distilledwater or left untreated. On days 3 and 5 animals were euthanized andentire wounds were harvested including the surrounding skin using an 8mm biopsy punch (Miltex). Wounds were fixed in 4% paraformaldehydeovernight at 4°, embedded in paraffin, and sectioned for analysis.

Hematoxylin and Eosin Staining (H&E):

All H&E staining was performed in the Histology Core Facility at theMedical University of South Carolina, Department of RegenerativeMedicine and Cell Biology. Briefly, sections were cleared in xylene,rehydrated through a series of graded alcohols, placed in Hematoxylinfollowed by acid alcohol. Samples were then placed in ammonia water,rinsed in ethanol and exposed to Eosin before dehydrating through gradedalcohols and clearing in xylene. Sections were mounted usingCytoseal-XYL (Richard-Allan Scientific). H&E sections were visualizedusing an Olympus BX40 microscope (4× objective lens, 0.13) and capturedusing an Olympus Camera (Model DP25) and DP2-BSW acquisition software.

Bacterial Inoculation, Tissue Gram Staining, Colon), Forming UnitQuantitation:

Male mice between 8-12 weeks were wounded as described above. Singlecolonies of Staphylococcus aureus (ATCC 25923) were picked and culturedovernight at 37° and adjusted to an absorbance of OD₆₀₀=0.53. One mL ofS. aureus was spun at 10,000 rpm, re-suspended in sterile PBS, and 15 μlwas used to innoculate each wound. sNAG membranes were applied to thetreated group thirty minutes post inoculation. Mice were euthanized onday 3 and 5 post wounding and wounds were harvested using an 8 mm biopsypunch. One wound per animal was fixed overnight in 4% paraformaldehydeat 4° C. and the other wound was cultured and plated on LB media withoutantibiotic for bacterial quantitation (see below). Wounds for tissuegram staining were embedded in paraffin and sectioned. Sections werecleared in xylene and rehydrated through a series of alcohol and werestained using a tissue gram stain (Sigma-Aldrich) by proceduresdescribed by the manufacturer.

For culturing, wound sections were placed in 0.5 ml bacterial media anincubated for 30 min at 37° C. while shaking. Colony forming units (CFU)were quantitated using a dilution series plated overnight at 37° C.Number of colonies per plate/per dilution were counted and CFU/ml werecalculated.

To determine CFU/ml from sNAG treated bacterial cultures, S. aureuscultures in solution were treated with varying concentrations of sNAG(10 μl and 20 μl of 10.8 mg/ml sNAG) for three hours. Cultures were thenplated overnight at 37° and CFU/ml were determined.

β-Defensin 3 Peptide Application:

Three test concentrations (1.0 μM, 2.5 μM, 5.0 μM) of biologicallyactive human β-defensin 3 peptide (Peptide Institute, Inc.) were testedfor their effect on bacterial growth in the infected wound healing modeldescribed above. Each concentration negatively affected bacterial growthso the lowest concentration was chosen for analyses. After each woundwas infected with S. aureus, 10 ul of peptide was applied. After threedays, wounds were harvested, embedded for sectioning and gram staining,or cultured for CFU/ml quantitation as described above.

β-Defensin 3 Antibody Blockade:

Wild Type male mice were wounded and infected with 15 ul of S. aureus asdescribed above. After inoculation, one wound was treated with 0.2 ug/mLof β-defensin 3 antibody (Santa Cruz) while the other was treated with0.2 ug/mL of normal goat IgG control antibody (Santa Cruz). sNAGmembranes were applied to all mice after antibody treatment on day 0.Antibody was applied every 24 hours. Mice were euthanized on day 3 andwounds were harvested using an 8 mm biopsy punch. Wounds were fixedovernight in 4% paraformaldehyde at 4° C., embedded in paraffin,sectioned, and analyzed using tissue gram stain. CFU/ml quantitation wasperformed from wounds harvested on day 3 as described above.

Immunofluorescence, Microscopy:

Paraffin embedded tissue sections were rehydrated through xylene and aseries of graded alcohols. Sections were treated with 0.01% Triton-X100and subjected to antigen retrieval using antigen unmasking solution(Vector Laboratories) in a pressure cooker for 5 min and allowed tocool. Skin sections were labeled with β-defensin 3 goat polyclonalantibody (Santa Cruz), involucrin rabbit polyclonal antibody (SantaCruz), and TO-PRO 3-iodide (Molecular Probes). Sections were incubatedin primary antibody overnight at 4° and appropriate secondaryimmunofluorescent antibodies (Invitrogen) for 1 hour at roomtemperature. Control sections for each antibody were stained withoutprimary antibody. Tissue sections were visualized using an OlympusFluroView laser scanning confocal microscope (Model IX70) and capturedat ambient temperature using an Olympus camera (Model FV5-ZM) andFluoview 5.0 acquisition software. All tissue sections were imaged using60× oil immersion lens (Olympus Immersion Oil)

HUVECs were either serum starved or treated with sNAG for 5 hours inculture and stained with antibodies directed against α-defensin 5(FITC), β-defensin 3 (Texas Red), or TOPRO 3 (Blue). Images were takenusing immunofluorescent microscopy. Cell culture defensin expression wasvisualized using a Zeiss Axiovert 100M confocal microscope and wascaptured at ambient temperature, using water as the medium, using LSM510 camera (Zeiss Fluor 63xW/1.2A objective).

Western Blot Analysis:

Endothelial cells were serum starved prior to stimulation with sNAG (50μl/ml) for a given time course. Cells were then lysed and subjected toWestern blot analysis. The antibodies used for Western blot analysis areas follows: anti-p85 subunit of PI3K and phosphospecific Akt antibody(Cell Signaling Technologies).

6.2.2 Results

6.2.2.1. Keratinocytes and Endothelial Cells Express and SecreteDefensins when Stimulated with sNAG

This example demonstrates that sNAG treatment modulates the expressionof defensins, small anti-microbial peptides that are part of the innateimmune response.

To investigate the affect of sNAG treatment on defensin expression invitro, primary human umbilical vein endothelial cells in culture wereused. Endothelial cells express both α-type and β-type defensins whenstimulated with sNAG. As shown in FIG. 7A endothelial cells treated withsNAG show an up-regulation of β-defensin 3 and α-defensin 1 mRNAexpression within 1 hour of stimulation. Similar up-regulation ofα-defensin 4 and 5 by sNAG treatment was also observed (data not shown).Custom gene arrays containing over 25 different defensin genes were usedto confirm the expression of the α-type defensins in primary endothelialcells and the β-type defensins in keratinocytes. sNAG stimulation ofendothelial cells was shown to increase the expression specifically ofα-defensins 1, 4 and 5 and β-defensin 3. Additionally, sNAG stimulationof human keratinocytes increased expression of β-defensin like genes,several of which are listed in Table 1. These findings suggest that atleast three α-defensin genes and β-defensin 3 are expressed in primaryendothelial cells and multiple β-defensin genes are expressed in primarykeratinocytes in response to sNAG stimulation.

TABLE I Gene array analysis reveals numerous defensin genes upregulatedby sNAG Fold Keratino- Fold HUVEC Gene Name Change cyte Gene Name Changeα-defensin 1 +1.36 β-defensin 1 +1.4 α-defensin 4 +2.74 β-defensin 126+1.73 α-defensin 5 +2.46 β-defensin +2.55 105B β-defensin 1 +2.19β-defensin 123 +1.65 β-defensin 4 +3.06 β-defensin 129 +1.46

To test whether the sNAG-dependent defensin expression also occurred onthe protein level, sNAG stimulated endothelial cells were subjected toimmunofluorescence using antibodies directed against both α and βdefensins. As shown in FIG. 7B, both β-defensin 3 and α-defensin 5 areup-regulated upon sNAG stimulation in this cell type. However,stimulation of primary human keratinocytes (HaCat) with sNAG did notcause increased expression of α-defensin but does cause an increase inthe expression of β-defensin 3 (FIG. 7C). Taken together, theseexperiments suggest that sNAG stimulation results in an up-regulation ofdefensin peptides in both primary keratinocytes and primary endothelialcells.

6.2.2.2. sNAG-Dependent Defensin Expression Requires Akt1

Previously published data show that sNAG stimulation of primaryendothelial cells results in increased integrin activation, Ets1expression and MAP kinase activation. (Vournmakis, J. N., et al., 2008,J Vase Res. 45(3):222-32.) Findings position Akt1 upstream of Ets1 inendothelial cells and in Drosophila. (Lavenburg, K. R., et al., 2003,FASEB J. 17(15): 2278-80.) To begin to determine the signaling pathwayresponsible for the expression of defensins, endothelial cells wereserum starved and pre-treated with pharmacological inhibitors directedagainst PI3K (wortmannin) or MAP kinase (PD098059) prior to sNAGstimulation. Quantitative real time PCR analysis shows that α-defensin 1mRNA levels are greatly diminished after inhibition of either thePI3K/Akt pathway or the MAP kinase pathway (FIG. 8A). RT-PCR analysis ofβ-defensin 3 also shows that levels are decreased by the inhibition ofthese pathways as well (FIG. 8B). sNAG treatment of endothelial cellsfor a short time course leads to phosphorylation of Akt1, a standardindicator of its activation (FIG. 8C). To confirm that Akt1 is indeedrequired for defensin expression, lentiviral delivery of shRNA directedagainst Akt1 was used. Quantitative RT-PCR of serum starved endothelialcells infected with scrambled (SCR) control or Akt1 shRNA followed withsNAG treatment confirms that Akt1 expression is required forsNAG-dependent α-defensin expression (FIG. 8D). Since β-defensins areknown to be expressed in epithelial cells, lentiviral delivery of shRNAdirected against Akt1 was used in human keratinocytes (HaCat). sNAGtreatment of serum starved keratinocytes infected with scrambled (SCR)control leads to a significant increase in β-defensin 3 expression thatis abrogated by Akt1 knockdown (FIG. 8E). These results illustrate thatsNAG treatment activates Akt1 in endothelial cells and strongly suggestthat sNAG-dependent defensin expression requires Akt1 in bothendothelial cells and keratinocytes.

6.2.2.3. sNAG Treatment of Cutaneous Wounds Increase Defensin ExpressionIn Vivo

To confirm the dependence of Akt1 for the expression of defensins invivo, wild type and Akt1 null animals were used in an excisional woundhealing model. Although most mammalian leukocytes express α-defensins(human, rabbit, rat, and hamster), mouse leukocytes do not expressα-defensins. Therefore, β-defensin expression in these mouse models wasfocused on. Treatment of cutaneous wounds with a dried form of sNAG, athin biodegradable membrane, for three days results in a statisticallysignificant increase in β-defensin 3 expression in keratinocytes of wildtype animals (FIG. 9A). Involucrin (Watt, F. M., 1983, J InvestDermatol. 81(1 Suppl): 100s-3s) staining (red) was used to mark thekeratinocyte cell layers and show that the expression of β-defensin 3 isconfined to the epidermal layer. To assess if sNAG-dependent defensinexpression is dependent on Akt1, a similar assay was performed using anAkt1 null animal model. Wounds from Akt1 null mice treated with sNAGmembranes show a markedly reduced induction of β-defensin 3 expression(FIG. 9A). To better visualize the epidermal layers that are expressingβ-defensin 3, FIG. 9B shows a representative image of a sNAG treatedwild type wound harvested on day 3. sNAG treatment of cutaneous woundsinduced β-defensin 3 expression mainly in the suprabasal layers of skin(FIG. 9B). Quantitative analyses shown in FIG. 9C shows an approximate5-fold increase in β-defensin 3 expression in sNAG treated wild typeanimals and that Akt1 is required for this increase.

6.2.2.4. sNAG Treatment Increases the Kinetics of Wound Closure in WTAnimals

Previous results have shown an increased kinetics of wound closure indiabetic mouse models in response to sNAG treatment. sNAGs were testedfor a similar affect in wild type animals. Excisional wounds werecreated in wild type animals which were either treated with the membraneform of sNAG or left untreated. Tissue sections were taken at 1, 3 and 5days post wounding and subjected to H&E staining. As shown in FIG. 10,sNAG treatment of wild type wounds results in complete closure, asvisualized by the solid line, at day 3 post wounding. This occurs twodays earlier than in the control wounds. Akt1 null animals display adelay in wound closure; these animals do not fully close the wound until7 days post wounding. The delay in wound closure in the Akt1 nullanimals is not rescued by sNAG treatment (data not shown). Thesefindings suggest that sNAG not only induces defensin expression but alsoincreases wound healing kinetics in wild type mice and may be a noveland effective therapeutic.

6.2.2.5. sNAG is an Effective Antimicrobial Against S. Aureus

Defensin peptides are known to possess antimicrobial properties that areactive against gram-positive and gram-negative bacteria. Since treatmentof endothelial cells with sNAG increases defensin expression (both α-and β-type) and treatment of cutaneous wounds with sNAG dramaticallyincreases β-defensin 3 expression in vivo, the antimicrobial efficacy ofsNAG treatment in bacterially infected wounds was assessed.

To determine if sNAG decreases bacterial load in cutaneous wounds, wildtype and Akt1 null animals were subjected to cutaneous wound healing,followed by infection with Staphylococcus aureus. Infected wounds wereeither treated with sNAG or left untreated for 3 and 5 days postinfection. As shown by the tissue gram staining in FIGS. 11A and 11B,wild type animals treated with sNAG show a significant reduction in grampositive staining by day 5 post wounding as compared with untreatedwounds. In contrast, gram stained tissue derived from untreated woundsin Akt1 null animals at 5 days post wounding show an accumulation ofneutrophils which stain gram positive (FIG. 11B), indicating a potentiallack of bacterial clearance in these animals that is not rescued by sNAGtreatment. These findings suggest that Akt1 null animals have a defectin immune clearance mechanisms which is not rescued by sNAG treatment.

To quantitate sNAG-specific bacterial changes in colony forming units(CFU), infected wounds from both wild type and Akt1 null mice eithersNAG treated or untreated were harvested and cultured. As shown in FIG.11C, at 5 days post wounding bacterial number is markedly reduced(10-fold) in wild type animals treated with sNAG. However, although thenumber of bacteria detected in the Akt1 null animals is reduced incomparison to wild type, sNAG treatment had a little effect on absolutebacterial number in the Akt1 null animals. At 3 days post-infection(FIG. 11D), there is a similar 10-fold decrease in CFU in sNAG treatedwild type mice as compared to untreated controls. The sNAG treated Akt1null animals show a 2-fold decrease in CFU as compared to untreated Akt1null animals. In general, the Akt1 null animals have a lower bacterialload per wound which may be reflective of an Akt1-dependent effect onother processes in addition to defensin expression. These findingssuggest that sNAG treatment results in a marked reduction in bacterialload in infected cutaneous wounds in wild type mice but not in Akt1 nullmice, suggesting the possibility that defensins are mediating theanti-bacterial response.

To show that the antibacterial effect of sNAG treatment is not due to adirect effect of the nanofibers on bacterial growth or on theirsurvival, S. aureus bacterial cultures were treated in solution withdifferent amounts of sNAG, for 3 hours and colony forming units weredetermined. As shown in FIG. 11E, sNAG treatment had no direct effect onthe growth of S. aureus, indicating that sNAG is not directly inhibitingbacterial growth and may then be working via the up-regulation ofdefensins.

6.2.2.6. Application of Defensin Peptide Mimics the sNAG AntibacterialEffect

To determine whether addition of defensin peptide can block bacterialinfection similarly to that shown for sNAG treatment, wild type micewere wounded and inoculated with S. aureus as described above and thentreated with biologically active human β-defensin 3 peptide (1.0 μm) forthree days. Tissue biopsies were stained using a tissue gram stain andCFU was quantitated. FIG. 11 F-G shows the results of these experiments.Infected mice treated with β-defensin 3 peptide have a decreasedbacterial load, an approximate 7.5 fold decrease in viable bacteria(FIG. 11G), similar to that shown in wild type mice treated with sNAG.

One of the mechanisms by which defensin expression is induced is throughstimulation by bacterial LPS, possibly through the activation of Tolllike receptors. (Selsted, M. E. and A. J. Ouellette, 2005, Nat Immunol.6(6):551-7.) To test whether bacterial infection alone is able to induceβ-defensin expression within the time periods tested, expression ofβ-defensin was assessed in infected wounds from wild type animals afterthree days post wounding. As shown in FIG. 12A, bacterial infectionalone does not induce the expression of β-defensin within 3 days ofinfection, as is shown with sNAG treatment. However, in wild typeanimals, sNAG treatment of infected wounds causes approximate 3- to5-fold increase in the expression of β-defensin within a similar timeperiod (FIG. 12B). These findings suggest that sNAG treatment rapidlyinduces the expression of defensin expression resulting in markedbacterial clearance in S. aureus infected wounds.

6.2.2.7. Antibodies Directed Against β-Defensin 3 Block theAntibacterial Effect of sNAG

Since defensins are secreted proteins, the inventors hypothesized thatantibodies directed against β-defensin 3 may be able to block theantibacterial activities. To test this hypothesis, wounds were created,infected with S. aureus and treated with sNAG as described above. Thewounds were either treated with a β-defensin 3 antibody or an isotypecontrol; one application each day for three days. Wound sections wereobtained and stained for gram positive bacteria. As shown in FIG. 13A,sections derived from wounds treated with β-defensin antibody have moregram positive bacteria than those treated with isotype controlantibodies. Each section shown was derived from the wound area directlyunder the scab. Quantitation of CFU in these wounds shows thatneutralization of β-defensin 3 prior to sNAG treatment in S. aureusinfected wounds results in a significant increase in bacteria. Animalsthat were treated with an IgG isotype control show an approximate 5-foldreduction in viable bacteria (FIG. 13B). Taken together, these resultssuggest that sNAG treatment not only results in the increased kineticsof wound healing but also promotes an endogenous anti-bacterial responseand supports the use of this nanofiber as novel therapy to enhance woundhealing while concurrently decreasing wound infection.

6.2.3 Conclusions

The findings presented here demonstrate that a marine diatom derivednanofiber, sNAG, may be used as a novel and effective method to enhancewound healing while concurrently decreasing wound infection. The datademonstrates that this FDA approved material, which is presently usedfor hemostasis, stimulates the expression of both α-type and β-typedefensins in primary endothelial cells and an up-regulation of theβ-type in primary keratinocytes.

Defensins are an essential component of the innate immune system. Thesepeptides possess anti-microbial properties that are active againstgram-positive and negative bacteria, fungi, and many viruses. Defensinsare small (3-4 kDa), cysteine-rich cationic peptides found in mammals,insects, and plants that are classified into different families (α, β,and θ) based on their pattern of disulfide bonding. α-defensins arethought to be specific to neutrophils, are found in very highconcentrations (comprising approximately 5-7% of the total cellularprotein) (Ganz, T. and R. I. Lehrer, 1994, Curr Opin Immunol.6(4):584-9), and are secreted during anti-microbial responses (Ganz, T.,1987, Infect Immun. 55(3):568-71). It has also been shown that rabbitalveolar macrophages possess α-defensins in levels comparable to rabbitneutrophils. (Ganz, T., et al., 1989, J Immunol. 143(4):1358-65.)β-defensins are found in epithelial cell types such as keratinocytes,mucosal epithelial cells (Harder, J., et al., 1997, Nature387(6636):861; and Harder. J., et al., 2001. J Biol Chem.276(8):5707-13), oral cavity tissues and salivary secretions (Mathews,M., et al., 1999, Infect Immun. 67(6):2740-5), and kidney where they canbe up-regulated in response to infectious or inflammatory stimuli (Ganz,T. and R. I. Lehrer, 1994, Curr Opin Immunol. 6(4):584-9). Humanβ-defensin 1 (hDEFB1) is one of the most important antimicrobialpeptides in epithelial tissues. Defensin expression and secretion couldbe extremely important for creating wound therapeutics. Theanti-microbial action by defensins is considered part of innate immunityand is non-specific and broad spectrum. Therefore acquired bacterialresistance, as seen with the overuse of antibiotics, is not an issue.

The data presented here also demonstrate that both in vitro and in vivoAkt1 is required for defensin expression. sNAG treatment decreases Staphaureus infection of cutaneous wounds in wild type control animals butnot in similarly treated Akt1 null animals. It is also important to notethat sNAG stimulation of wild type cutaneous wounds results in anincreased kinetics of wound closure. Antibody blockade of β-defensinresults in a reduction in the sNAG-antibacterial activity. Takentogether these findings suggest a central role for Akt1 in theregulation of defensin expression that is responsible for the clearanceof bacterial infection and that sNAG treatment activates these pathwaysin wild type animals.

The data that suggests that sNAG treatment of infected wounds coulddrastically decrease bacterial load in patients, at least in part, bythe induction of defensin expression. Staphylococcus aureus is abacterium frequently found colonizing the skin and in the nose. It isstill a common cause of nosocomial infections, often causingpostsurgical wound infections. S. aureus infections in hospitals haveplagued healthcare workers for years and the widespread usage ofantibiotics for treatment has lead to antibiotic resistant strains. Thedata presented herein shows that treatment of Staph infected wounds withsNAG dramatically decreased the bacterial load. For example, the lack ofdark purple gram staining in the treated WT mice in FIGS. 11A and 11Bindicates that the S. aureus infection has been cleared from thesewounds. Both the in vitro and in vivo data provides strong evidence forthe use of Taliderm/sNAG in the treatment of wounds to decreasebacterial infection and therefore enhance wound healing.

Control experiments indicate that the antibacterial effect of sNAG isnot due to a direct interaction of the material with the bacteria but isdue to downstream affects such as the regulation of defensins by Akt1activation. It is widely accepted that defensins are important playersin innate immunity and function in antimicrobial activities. Most of theevidence for their function is the direct killing of bacteria by invitro mixing experiments with purified defensin peptides (Selsted, M. E.and A. J. Ouellette, 2005, Nat Immunol. 6(6):551-7) or in similarexperiments as shown in FIG. 11 with direct application of the purifiedactive peptide. The data here show that an induction of defensinexpression in wild type animals using a topical application of sNAGresults in an antibacterial response. It has recently been shown thattransgenic mouse models expressing the human defensin 5 gene areresistant to S. typhimurium, an infection that results in death ofwild-type animals (Salzman, N. H., et al., 2003, Nature 422(6931):522-6)again suggesting the importance of defensins in the regulation of theantimicrobial response.

It has been accepted that the α-subtype of defensins are specificallyexpressed in neutrophils, whereas the β-type defensins are epithelial inorigin. β-type defensin expression induced in response to sNAG in humankeratinocytes both in culture and in the cutaneous wound healing modelwas detected. The in vivo data illustrates that β-defensin 3 is mainlyexpressed in the suprabasal layers after treatment with sNAG. This isconsistent with previous data which localized human β-defensin 2 to thespinous and granular layers of the skin. (Oren, A., et al., 2003, ExpMol Pathol. 74(2):180-2.) The skin is in constant contact with injuryand infection and functions not only as a mechanical barrier but alsomaintains the ability to mount an active defense against infection. Theexpression of β-defensin in the outer layers of skin supports their rolein cutaneous innate immunity. However, the data show that sNAGspecifically stimulates the expression of three different α-defensins(1, 4 and 5) in endothelial cells. This is shown by RT-PCR, gene arrayanalysis, immunofluorescence and ELISA (data not shown). The interactionbetween endothelial cells and leukocytes in tissue repair is one of theinitial and most important steps in wound healing. The process ofextravasation of leukocytes from the vasculature is initiated bychemotactic factors, therefore; it is interesting that α-defensins areinduced by sNAG and may contribute to the necessaryneutrophil/endothelial cellular interactions. More recently, it has cometo light that defensins exhibit biological activities beyond theinhibition of microbial cells, including their contribution to theadaptive immune response by exhibiting chemotactic activity on dendritic(Hubert, P., et al., 2007, FASEB J. 21(11):2765-75) and T cells,monocytes, and macrophages (Garcia, J. R., et al., 2001, Cell TissueRes. 306(2):257-64) and keratinocytes (Niyonsaba, F., et al., 2007, JInvest Dermatol. 127(3):594-604). Previous work shows that human betadefensins 1 and 2 have the ability to chemoattract immature dendriticcells and T cells through the CC-chemokine receptor 6 (CCR6) (Yang, D.,et al., 1999, Science 286(5439):525-8), and that human beta defensin 2can chemoattract TNFα treated neutrophils via the CCR6 receptor(Niyonsaba, F., H. Ogawa, and I. Nagaoka, 2004, Immunology111(3):273-81). Human β-defensin 2 and 3 have also been shown to inducechemotaxis by interacting with CCR2, a receptor expressed onmacrophages, monocytes, and neutrophils. (Rohrl, J., et al., 2010, JImmunol, 2010.) Interestingly, the data show that sNAG treatment inducesboth α and β-defensin expression in endothelial cells. Taken together,the recent data suggest that defensins may mediate wound healing notonly by their antimicrobial properties, but also by being chemotacticfor other cell types necessary for proper healing. However, applicationof β-defensin 3 alone did not result in an increase in wound closure(data not shown) implying that topical application of a single defensindoes not sustain the cellular interactions required for increased chemoattraction, cellular recruitment and wound closure.

The in vivo data using both wild type and Akt1 knockout animals confirmsthe requirement for Akt1 in sNAG-induced β-defensin 3 expression. Sincemouse leukocytes do not express α-defensins like most other mammalianleukocytes (Ganz, T., 2004, C R Biol. 327(6):539-49) in vivo α-defensinstaining of infiltrating immune cells was not possible. Treatment ofairway epithelial cells in vitro with alpha defensins 1-3 causes a doseand time-dependent increased cell migration that requires activation ofPI3K and MAPK pathways. (Aarbiou, J., et al., 2004, Am J Respir Cell MolBiol. 30(2):193-201.) sNAG stimulation of endothelial cells has beenshown to result in the activation of MAPK (Vournakis, J. N., et al.,2008, J Vase Res. 45(3):222-32) and in data presented here,pharmacological inhibition of MEK also inhibits the expression of thedefensins in vitro. These findings suggest that both pathways impinge onthe regulation of defensin expression by sNAG, however, Akt1 ablationresults in a marked reduction of its expression both in vitro and invivo. In myeloid cells, β-defensin 1 expression is controlled at thelevel of transcription, in part, by the Ets-family member PU.1. (Yaneva,M., et al., 2006, J Immunol. 176(11):6906-17; and Ma, Y., Q. Su, and P.Tempst, 1998, J Biol Chem. 273(15):8727-40.) PU.1 is a downstream targetof Akt1 in the B-cell lineage. (Rieske, P. and J. M. Pongubala, 2001, JBiol Chem. 276(11):8460-8.) In primary endothelial cells it has beenshown that Akt1 is upstream of Ets1 both in vitro and in vivo duringDrosophila tracheal development. (Lavenburg, K. R., et al., 2003, FASEBJ. 17(15):2278-80.) sNAG stimulation of endothelial cells results inincreased expression of Ets1 (probably through Akt1) which is requiredfor the migration of endothelial cells. (Vournakis, J. N., et al., 2008,J Vasc Res. 45(3):222-32.)

Thus far, sNAG treatment has resulted in a series of downstreamactivities; hemostasis, cell migration, cell proliferation, increasedwound closure, and as described here, stimulation of the innate immuneresponse resulting in anti-bacterial functions.

Given the dramatic increase of diabetic patients within the populationwho present with chronic wounds and complications due to woundinfection, new clinical treatments are in high demand. Here, marinederived pGlcNAc nanofibers are described that not only increase thekinetics of wound healing but act to stimulate innate immunity thusproviding anti-bacterial activity. The obvious importance of theseobservations is the application to nosocomial infections. Of thenosocomial infections, surgical wound infections predominate; withstatistics showing up to 8% of all surgical patients. The direct cost ofthese types of infections is approximately 4.5 billion dollars per year.Given that defensins are part of the innate immune system, activation ofthese pathways will preclude the generation of resistant organisms aswell as allow for the antibiotic-independent clearance of bacterialinfection. Use of sNAG in a hospital setting would defray much of thecost and markedly reduce the production of antibiotic resistant species.Taken together, these findings suggest that these marine derived pGlcNAcnanofibers will be highly beneficial in the clinical arena.

6.3 Example 3 sNAG is an Effective Antimicrobial Against Pseudomonasaeruginosa

This example demonstrates that sNAG nanofibers have an anti-bacterialeffect against Pseudomonas aeruginosa in vivo.

Materials and Methods:

Wild Type C57Bl/6 male mice between 8-12 weeks old were wounded createdusing a 4 mm biopsy punch (Miltex), to create two identical wounds oneach flank. Mice were anesthetized using an O₂/Isoflurane vaporizinganesthesia machine (VetEquip, Inc.). Isoflurane was used at 4% forinduction; 2% for surgery. Prior to surgery hair was removed byPseudomonas aeruginosa were picked and cultured overnight at 37° andadjusted to an absorbance of OD₆₀₀=0.53. Each wound was inoculated with1.5×10⁹ cfu/wound of P. aeruginosa. After 30 minutes post inoculation,wounds were either treated with sNAG membrane moistened with distilledwater (test group, n=6) or left untreated (control group, n=6). On day 3animals were euthanized and entire wounds were harvested including thesurrounding skin using an 8 mm biopsy punch (Miltex). One wound peranimal was fixed overnight in 4% paraformaldehyde at 4° C.°, embedded inparaffin, and sectioned for analysis, and the other wound was culturedand plated on LB media without antibiotic for bacterial quantitation.For culturing, wound sections were placed in 0.5 ml bacterial media anincubated for 30 min at 37° C. while shaking. Colony forming units (CFU)were quantitated using a dilution series plated overnight at 37° C.Number of colonies per plate/per dilution were counted and CFU/ml werecalculated.

Results:

The efficacy of sNAG treatment of wounds infected with gram negativebacteria was assessed. As shown in FIG. 14, at 3 days post infectionbacterial number is markedly reduced (more than 2 fold) in animalstreated with sNAG in comparison to untreated animals. These findingssuggest that sNAG treatment results in a marked reduction in bacterialload of gram negative bacteria, and specifically P. aeruginosa, ininfected cutaneous wounds (in addition to reduction in bacterial load ofgram positive bacteria shown in Example 2).

6.4 Example 4 sNAG Nanofibers Upregulate Expression of a Number ofDefensins and Toll Receptor Genes

This example demonstrates that a number of defensins and Toll-likereceptors are up-regulated by sNAG treatment of human endothelial cells.

Materials and Methods:

Human Chip probes were printed on epoxy slides. HUVEC cells werecultured as described in section 6.2, and treated with sNAG nanofibers(“sNAG”) for 5 hours. RNA was extracted with RNAsol (Teltest, Inc.)following manufacturer's instructions, amplified using Amino AllylMessageAMP™ II aRNA amplification kit (Applied Biosystems), and labeled.The slides were prepared for hybridization with aRNA by soaking inblocking solution (Sigma Tris-buffered saline pH8.0, in 1000 ml dH₂O, 1%BSAw/v, NaN₃ to 0.05%) at RT O/N, then rinsed and dried. Samplescontaining labeled target aRNA from sNAG-treated cells were hybridizedwith the slides (65 ul/slide; denatured at 95° C. for 5 min; hybridizedfor 48 hours at 37° C. in 0.1% SDS and 5×SSC and 1% BSA), rinsed anddried. The slides were scanned and hybridization detected usingPerkin-Elmer Scan Array equipment and ScanArray Express software V3.0,updated. To identify up-regulated genes, microarray data was analyzedusing Agilent GeneSpring GX v. 11 Bioinformation Data Analysis.

Genes of interest analyzed: IL-1, CEACAM3, SPAG11, defensins(“DEFA”=α-defensin, and “DEFB”=β-defensin); Toll-like receptors (“TLR”),SIGIRR (Single IG IL-1-related receptor), and TRAF6 (TNF receptorassociated factor 6). Positive controls: 1433Z(Tyrosine-3-monohydrogenase/tryptophan 5 monohydrogenase actitionprotein); GAPD (glyceraldehydes-3-phosphate dehydrogenase); RPL13A(Ribosomal protein L13a); UBC (Ubiquitin C); ACTB (Actin B).

Results:

Results of the microarray gene chip analyses and Q-PCR validation ofmicroarray results are presented in Tables II-VI below. Using a customgene chip it was determined that a number of defensins and Toll-likereceptors are up-regulated by sNAG treatment of human endothelial cells.

Toll-like receptors (TLRs) are highly conserved receptors that recognizespecific molecular patterns of bacterial components leading toactivation of innate immunity. Interestingly, Drosophila lack anadaptive immune system but are still resistant to microbial infections.(Imler, J. L. and J. A. Hoffmann, 2000, Curr Opin Microbiol, 3(1):16-22.) This host defense is the result of an innate immune system thatprovides protection by synthesizing the antimicrobial peptides dToll and18-wheeler which are induced by TLRs. (Lemaitre, B., et al., 1996, Cell86(6):973-83; and Williams, M. J., et al., 1997, EMBO J.16(20):6120-30.) Recent work has also linked human defensin expressionto TLR activation. Human β-defensin 2 was shown to be induced in airwayepithelial cells in a TLR-2 dependent manner. (Hertz, C. J., et al.,2003, J Immunol. 171(12): p. 6820-6.) Toll-like receptor 4 has beenshown to mediate human β-defensin 2 inductions in response to Chlamydiapneumonia in monocytes. (Romano Carratelli, C., et al., 2009, FEMSImmunol Med Microbiol. 57(2):116-24.) Importantly, the PI3K/Akt pathwayis a key component in TLR signal transduction, controlling cellularresponses to pathogens. (Weichhart, T. and M. D. Saemann, 2008, AnnRheum Dis. 67 Suppl 3:iii70-4.) Since it is known that stimulation ofTLRs can lead to increased defensin synthesis, this work suggests thepotential for sNAG as a stimulator of innate immunity and bacterialclearance via the activation of Akt1.

TABLE II List of some genes up-regulated in response to sNAG stimulationGene Function IL-1 Pro-inflammatory cytokine involved in immune defenceCEACAM3 Cell adhesion molecule which directs phagocytosis of severalbacterial species SPAG11 β-defensin-3 like molecule that exhibitsantimicrobial properties Defensins A series of defensins that exhibitantimicrobial activity TLRs Toll-like receptors: important forstimulation of cellular responses toward infection FOLD GENELIGAND/FUNCTION INDUCTION TLR1 Triacyl lipopeptides from bacteria and7.6 mycobacteria TLR4 LPS, viral proteins, Hsp60 (Chlamydia) 5.064 TLR7synthetic compounds 3.271 TLR8 synthetic compounds 2.067 TRAF6Downstream signalling modulator 6.167 SIGRR IL-1 receptor related TLRmodulator 5.895

TABLE III Defensin Microarray Gene Expression (HUVEC Response to sNAG 10ug/ml 5 hours) HUVEC_10s_48h37C Gene Name [Oligo ID] normalized (Fold)D107A_HUMAN [H300005354] 4.2 (2.6 to 5.2) DEFA4 [H200000646] 4.2 (3.243to 4.946) DEFA5 [H200005803] 4.8 (3.664 to 6.123) DEFB1 [H200004191] 2.7(1.7 to 3.7) DEFB103A [H300008014] 9.8 (7.4 to 12.5) DEFB118[H200017001] 2.7 (1.502 to 4.779) DEFB119 [H300002796] 6.2 (4.68 to8.04) DEFB123 [H300009262] 8.9 (7.791 to 11.1) DEFB124 [H300001942] 3.8(1.6 to 5.1) DEFB126 [H200012496] 9.2 (8.286 to 10) DEFB129 [H300085026]5.2 (4.338 to 6.277) ACTB_HUMAN [H300006234] 6.8 (6.603 to 7.284) GAPD[H200007830] 16.9 (12.81 to 21.13) RPL13A [opHsV04TC000041] 9.4 (7.311to 12.01) UBC [H200014214] 7.2 (5.789 to 9.979) 1433Z_HUMAN 0.6 (0.4 to0.844) [opHsV04TC000038]

TABLE IV DEFCB3 Microarray Gene Validation (AB Prism 7000; sNAG (10ug/ml), HUVEC for 5 h) TaqMan Relative qPCR Fold Change Calculations(ABI method) Fold difference ΔCt = ΔΔCt = in DEFB3 DEFB3 − ΔCttreated −relative to Sample DEFB3 1433z 1443z ΔCt untreated untreated untreated37.41 ± 0.74 14.71 ± 0.26 22.7 ± 0.78 0.00 ± 0.78 1.4 (1.22 − 1.7) treated 40.30 ± 1.0  17.84 ± 0.07 22.46 ± 1.0  −0.24 ± 1.0  1.8 (1.24 −2.36)

TABLE V Toll-Like Receptors Microarray Gene Expression Gene Naree [OligoID] Fold Change SIGIRR [opHsV0400002471] 5.895 (3.916 to 7.926) TLR1[H300000701] 7.612 (3.796 to 11.33) TLR4 [H200007406] 5.064 (1.085 to10.66) TLR7 [H200008345] 3.271 (1.938 to 3.938) TLR7 [H300006695] 2.2(1.5 to 2.7) TLR8 [H200016915] 2.067 (1.8 to 2.2) TRAF6 [H200010465]6.167 (5.2 to 7) 1433Z_HUMAN [opHsV04TC000038] 0.573 (0.4 to 0.844)

TABLE VI Real Time Q-PCR Gene Validation of TLR1 & 4 (HUVEC, 10 ug/mlsNAG for 5 h) Reference Reference (1433z) (1433z) ΔC_(T) = ΔC_(T) sd =ΔΔCt = Fold Fold Target Target C_(T) C_(T) TargetC_(T) − (S_(target) ² +ΔCt_(test sample(treated)) − ΔΔCt sd = up down Fold Sample C_(T) aveC_(T) sd ave sd 1433zC_(T) S_(reference) )^(2 1/2)ΔCt_(Calibrator(untreated)) ΔCt sd 2^(−(ΔΔCt+sd)) 2^(−(ΔΔCt−sd)) aveTLR₁ untreated 31.12 1.2 17.84 0.34 13.28 1.25 0 1.25 0.42 2.37 0.83treated 28.54 0.37 17.53 0.2 11.01 0.42 −2.27 0.42 3.60 6.46 5.03 TLR₄untreated 26.97 0.44 17.84 0.34 9.13 0.56 0 0.56 0.68 1.47 0.62 treated25.04 0.38 17.53 0.2 7.51 0.43 −1.62 0.43 2.28 4.14 3.21

6.5 Example 5 sNAG and Lone Fiber NAG Differ in their Gene ExpressionProfiles

This example demonstrates that sNAG nanofibers differ from long p-GlcNAcfibers in their effect on gene expression, and specifically in theireffect on expression of some of the defensins and Toll-like receptors.

Materials and Methods:

Human Defensin Chip probes (concentration: 20 uM, quantity 18-20,solvent: SSC based spotting buffer) were printed on epoxy slides usingstandard techniques. HUVEC and HaCat cells were cultured as described insection 6.2, and treated with either long fibers (“LNAG”) or sNAGnanofibers (“sNAG”), for 2 hours or 20 hours. RNA was extracted withRNAsol (Teltest, Inc.) following manufacturer's instructions, andamplified using Amino Allyl MessageAMP™ II aRNA amplification kit(Applied Biosystems). During RNA amplification, aRNA from cells treatedwith LNAG and aRNA from cells treated with sNAG was differentiallylabeled with Cy3 or Cy5 fluorescent dyes. The slides were prepared forhybridization with aRNA by soaking in blocking solution (SigmaTris-buffered saline pH8.0, in 1000 ml dH₂O, 1% BSAw/v, NaN₃ to 0.05%)at RT O/N, then rinsed and dried. Samples containing equal amounts ofdifferentially labeled target aRNA from LNAG and sNAG-treated cells weremixed, hybridized with the slides (65 ul/slide; denatured at 95° C. for5 min; hybridized for 48 hours at 37° C. in 0.1% SDS and 5×SSC and 1%BSA), rinsed and dried. The following exemplary graphs in Table VIIillustrate experimental set up:

TABLE VII Labeling of aRNA 20 ug total aRNA for labeled labeled ng/ul260/280 label dye conc. aRNA Name (100 ul) nm (ul) used Pmol/ul 260/280(20 ul) HaCat_e14d3_ctr 897.42 2.09 22.29 cy3 851.58 1.34 17031.6HaCat_e14d3_LNAG100 1339.08 2.07 14.94 cy5 687.01 1.87 13740.2HaCat_e14d3_sNAG100 1515.62 2.05 13.20 cy5 519.15 1.93 10383HUVEC_e18d4_ctr 1656.37 2.05 12.07 cy3 529.11 1.88 19577.07 37 ulHUVEC_e18d4_LNAG100 1078.63 2.07 18.54 cy5 760.26 1.9 15205.2HUVEC_e18d4_sNAG100 1447.87 2.06 13.81 cy5 617.57 1.84 12351.4 LabeledaRNA Hybridization Total aRNA Total 10% chip ID aRNA/slide conc. vol.SDS 20 × SSC D H₂O Total 37 C Chip ID Sample ID (ng) (ng/ul) (μl) (μl)(μl) (μl) Vol (ml) 48 h 37 C 48 h HaCat Actr 800 851.58 0.9 2 50 125.9200 D1038 D1034 ALNAG100 (Mix 1) 800 687.01 1.2 0 0 Actr 800 851.58 0.92 50 125.5 200 D1037 D1033 AsNAG100 (Mix 2) 800 519.15 1.5 0 0 HUVECVCtr 800 529.11 1.5 2 50 125.4 200 D1036 D1032 VLNAG100 (Mix 3) 800760.26 1.1 Vctr 800 529.11 1.5 2 50 125.2 200 D1035 D1031 VsNAG100(Mix4) 800 617.57 1.3

The slides were scanned and hybridization detected using Perkin-ElmerScan Array equipment and ScanArray Express software V3.0, updated. Foreach slide, Cy5, Cy3 and composite fluorescence was visualized. Toidentify up-regulated and down-regulated genes microarray data wasanalyzed using Agilent GeneSpring GX v.11 Bioinformation Data Analysis.Genes of interest analyzed: DEFA1, DEFA3, DEFA4, DEFA5, DEFA6, DEFB1,DEFB013A, DEFB104A, DEFB105B, DEFB108B, DEFB112, DEFB114, DEFB118,DEFB119, DEFB123, DEFB124, DEFB125, DEFB126, DEFB127, DEFB128, DEFB129,DEFB131, and DEFB4 (“DEFA”=α-defensin, and “DEFB”=β-defensin); TLR1,TLR10, TL2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR8 (“TLR”=Toll receptor);SIGIRR (Single IG IL-1-related receptor); IRAK2 (IL-1receptor-associated kinase 1); TRAF6 (TNF receptor associated factor 6);D106A (β-defensin 106), D107A (β-defensin 107). Negative controls: threerandom sequences (1, 2, 3). Positive controls: 1433Z(Tyrosine-3-monohydrogenase/tryptophan 5 monohydrogenase actitionprotein); GAPD (glyceraldehydes-3-phosphate dehydrogenase); RPL13A(Ribosomal protein L13a); UBC (Ubiquitin C); ACTB (Actin B).

Results:

Results of the microarray gene chip analyses are presented in TablesVIII and IX below. Table VIII shows gene expression in human umbilicalvein endothelial cells (“HUVEC”) after 2 h or 24 h exposure to eitherLNAG fibers or sNAG nanofibers. Table IX shows gene expression in humankeratinocyte cell line (HaCat) after 2 h or 24 h exposure to either LNAGfibers or sNAG nanofibers. The results demonstrate that gene expressionprofile induced by long poly-N-acetylglucosamine fibers (“LNAG”) differsfrom the gene expression profile induced by sNAG nanofibers (“sNAG”).Specifically, LNAG and sNAG differ in their effect on expression ofdefensin genes and Toll receptor genes.

TABLE VIII Microarray Defensin Gene Expression in Human Umbilical VeinEndothelial Cells (HUVEC), Fold Change [2 h, [2 h, [20 h, [20 h, NameLNAG] sNAG] Name LNAG] sNAG] 1433Z_HUMAN 0.039 0.329 1433Z_HUMAN −0.046−0.180 ACTB_HUMAN −0.140 0.032 ACTB_HUMAN 0.874 −0.413 D106A_HUMAN−1.376 −0.195 D106A_HUMAN 1.107 0.522 D107A_HUMAN 1.825 1.431D107A_HUMAN −1.007 0.372 DEFA1 0.407 −1.107 DEFA1 −0.333 0.384 DEFA30.000 0.528 DEFA3 1.195 −2.335 DEFA4 −1.007 −0.123 DEFA4 0.496 2.636DEFA5 −0.863 0.451 DEFA5 −0.287 −0.476 DEFA6 1.969 0.805 DEFA6 0.333−1.402 DEFB1 0.315 1.441 DEFB1 1.933 0.413 DEFB103A 1.426 1.486 DEFB103A0.628 1.348 DEFB104A 1.296 2.260 DEFB104A 1.543 0.344 DEFB105B 0.6160.667 DEFB105B 0.723 −0.162 DEFB108B 2.210 0.441 DEFB108B 0.351 1.895DEF8112 0.000 −0.528 DEFB112 −0.862 1.107 DEFB114 0.000 0.667 DEFB114−0.862 1.799 DEFB118 −0.142 0.631 DEFB118 0.456 0.577 DEFB119 0.1371.472 DEFB119 0.808 −1.530 DEFB123 1.664 1.814 DEFB123 0.390 −0.375DEFB124 1.242 1.533 DEFB124 1.113 1.357 DEFB125 1.169 1.969 DEFB1251.269 −2.053 DEFB126 −0.064 0.801 DEFB126 1.818 0.385 DEFB127 1.7230.000 DEFB127 0.000 1.085 DEFB128 1.602 −0.528 DEFB128 0.805 2.238DEFB129 1.528 0.407 DEFB129 1.936 −0.005 DEFB131 −0.333 0.636 DEFB131−0.723 −0.608 DEFB4 0.406 0.567 DEFB4 0.401 −0.190 GAPD 0.420 0.602 GAPD0.616 0.324 IRAK2 −0.035 1.106 IRAK2 1.084 0.984 RPL13A 0.671 1.329RPL13A 0.789 0.208 SIGIRR 0.358 1.481 SIGIRR 1.870 −0.050 TLR1 −0.1941.089 TLR1 0.196 −0.631 TLR10 0.000 −0.333 TLR10 −0.528 0.644 TLR2 0.6532.078 TLR2 1.848 4.494 TLR3 −0.528 −0.333 TLR3 −1.484 −1.361 TLR4 0.6132.073 TLR4 2.616 0.634 TLR5 1.723 1.181 TLR5 0.723 −0.417 TLR6 1.3330.528 TLR6 0.246 −0.482 TLR7 1.839 1.274 TLR7 −0.160 0.199 TLR8 −0.0330.843 TLR8 −0.371 1.219 TRAF6 1.569 0.472 TRAF6 0.731 3.266 UBC −0.2850.072 UBC −0.009 −0.265

TABLE IX Microarray Defensin Gene Expression in Human Keratinocyte CellLine (HaCat), Fold Change Name 2 h, LNAG 2 h, sNAG Name 20 h, LNAG 20 h,sNAG 1433Z 0.255 −0.282 1433Z 0.000 0.205 GAPD 0.041 −0.191 GAPD 0.0000.378 RPL13A −0.532 0.698 RPL13A 0.000 −1.187 UBC 0.136 −0.065 UBC 0.834−0.023 ACTB 0.130 0.447 ACTB 0.333 0.988 Negative 0.000 0.000 Negative0.000 0.000 Control Control Negative 0.000 0.000 Negative 0.000 0.000Control Control Negative 0.000 0.000 Negative 0.000 0.000 ControlControl DEFB1 −0.647 1.390 DEFB1 −0.333 −0.426 DEFB126 0.348 1.737DEFB126 1.000 0.744 DEFB129 0.382 1.464 DEFB129 −0.528 −0.931

6.6 Example 6 Effect of Irradiation on sNAG Membranes

Method of Preparation of sNAG Membrane.

The sNAG membrane is derived form microalgal pGlcNAc fibers produced aspreviously described (see Vournakis et al. U.S. Pat. Nos. 5,623,064; and5,624,679, the content of each of which is incorporated herein byreference in its entirety). Briefly, microalgae were cultured in uniquebioreactor conditions using a defined growth media. Following theharvest of microalgae from high-density cultures, fibers were isolatedvia a stepwise separation and purification process resulting in batchesof pure fibers suspended in water for injections (wfi). Fibers wereformulated into patches by concentration and oven drying, and werepackaged and sterilized by gamma-irradiation. Fiber dimensions average20-50 nm×1-2 nm×˜100 μm. Batches of fibers were individually qualitycontrolled using chemical and physical test parameters, and each batchmet strict purity criteria prior to release. Final batches were requiredto be substantially free of proteins, metal ions, and other components.The fibers were then shortened by irradiation to produce sNAG membranes.Briefly, the starting material contained 60 g of pGlcNAc slurry at aconcentration of 1 mg/mL. The concentration of the pGlcNAc slurry wasconfirmed by filtering 5 mL into a 0.2 um filter. 15 L of pGlcNAc slurrycontaining 15 g pGlcNAc was filtered until formation of a wet cake. Thewake cake was then transferred into a foil pouch, which is a gammaradiation compatible container, and subjected to 200 kGy gammaradiation. Other irradiation conditions were tested for their effects onpGlcNAc compositions, as reflected in FIG. 15A.

Effect of Irradiation on pGlcNAc Membranes.

While irradiation reduces the molecular weight of pGlcNAc, irradiationdid not disturb the microstructure of the fibers. pGlcNAc was irradiatedunder different conditions: as a dry, lyophilized material; as a drymembrane; as a concentrated slurry (30:70 weight by volume); and as adilute slurry (5 mg/ml). A suitable molecular weight reduction (to amolecular weight of 500,000-1,000,000 daltons) was achieved at anirradiation dose of 1,000 kgy for dry polymer, and 200 kgy for wetpolymer (FIG. 15A).

The chemical and physical structure of the fibers was maintainedthroughout irradiation as verified by infrared (IR) spectrum (FIG. 15B),elemental assay, and scanning electron microscopes (SEMs) analysis.Microscopic observation of irradiated fibers showed a decrease in theparticle length (FIGS. 15C and 15D). The majority of the fibers are lessthan about 15 μm in length, with an average length of about 4 um.

6.7 Example 7 sNAG Nanofibers and Lone Form p-GlcNAc Fibers Differ intheir Effects on Metabolic Rate and Serum Deprivation of Umbilical CordVein Endothelial Cells

Materials and Methods.

Pooled, multiple-donor human umbilical cord vein endothelial cells (EC)(Cambrex) were maintained at 37° C. with 5% CO₂ in endothelial basalmedium 2 (Cambrex) supplemented with EC growth medium 2 SingleQuots asdescribed by Cambrex procedures. Serum starvation was performed at80-90% confluency in RPMI-1640 supplemented with 0.1% fetal calf serum(Gibco BRL) for 24 h followed by stimulation with VEGF 165 (20 ng/ml,R&D Systems) or with highly purified pGlcNAc nanofibers or sNAGnanofibers in sterile water (provided by Marine Polymer Technologies,Inc., Danvers, Mass., USA) with the amounts indicated in the figuredescriptions. For cellular proliferation/viability assessment, 2different assays were used: trypan blue exclusion by direct cell countsusing a hemacytometer and an MTT[3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay inprocedures described by the manufacturer (Promega).

Results—pGlcNAc:

pGlcNAc Did not Affect Metabolic Rate.

As shown in FIG. 16, pGlcNAc did not result in a higher metabolic rateas measured by MTT assays, indicating that this polymeric material wasnot causing marked increases in cellular proliferation.

pGlcNAc Protected EC from Cell Death Induced by Serum Starvation.

To test if pGlcNAc fibers had a direct effect on EC, serum-starved ECcells were treated with VEGF or with different concentrations of pGlcNAcfibers. As shown in FIG. 17 at 48 h and 72 h after serum starvation, ascompared with the total number of cells plated (control), there wasabout 2-fold reduction in the number of cells after 48 h or 72 h. At 48h, this decrease in cell number was rescued by the addition of VEGF orby the addition of pGlcNAc fibers at either 50 or 100 μg/ml. At 72 h,the decrease in cell number was rescued by the addition of VEGF orlargely rescued by the addition of pGlcNAc fibers at 100 μg/ml. Theseresults indicated that like VEGF, pGlcNAc fiber treatment prevented celldeath induced by serum deprivation.

Results—sNAG:

sNAG Induced Marked Increase in Metabolic Rate.

As measured by MTT assays, sNAG at 50, 100 or 200 μg/ml resulted in ahigher metabolic rate of EC than VEGF (FIG. 18).

sNAG Did not Protect EC from Cell Death Induced by Serum Deprivation.

To test if sNAG fibers had a direct effect on EC, serum-starved EC cellswere treated with VEGF or with different concentrations of sNAG fibers.As shown in FIG. 19, at 48 h after serum starvation, as compared withthe total number of cells plated (control), there was about 2-foldreduction in the number of cells. This decrease in cell number wasrescued by the addition of VEGF but not by the addition of sNAG fibersat 50, 100 or 200 μg/ml. These results indicated that not like VEGF,sNAG fiber treatment did not prevent cell death induced by serumdeprivation.

Conclusion:

The above results demonstrate that sNAG, unlike long form pGlcNAc,increases the metabolic rate of serum-starved EC in a MTT assay and doesnot rescue apoptosis of serum-starved EC in a trypan blue exclusiontest.

6.8 Example 8 Preclinical Testing of sNAG

6.8.1 Test Article

A test article comprising sNAG produced as previously described inSection 6.2.1 supra. was utilized. The test article was supplied sterileby Marine Polymer Technologies, Inc.

6.8.2 Biocompatibility Testing—L929 MEM Elusion Test—ISO 10993-5

Biocompatibility of the test article was tested in mouse fibroblast L929mammalian cells. No biological reactivity (Grade 0) was observed in theL929 cells at 48 hours, post exposure to the test article. The observedcellular response obtained from the positive control article (Grade 4)and negative control article (Grade 0) confirmed the suitability of thetest system. Based on the criteria of the protocol, the test article isconsidered non-toxic and meets the requirements of the Elution Test,International Organization for Standardization (ISO) 10993-5 guidelines.See Table X below.

TABLE X REACTIVITY GRADES Controls Test Article Medium Negative PositiveTime A B C A B C A B C A B C  0 Hours 0 0 0 0 0 0 0 0 0 0 0 0 24 Hours 00 0 0 0 0 0 0 0 3 3 3 48 Hours 0 0 0 0 0 0 0 0 0 4 4 4 Grade ReactivityDescription of Reactivity Zone 0 None Discrete intracytoplasmicgranules; no cell lysis 1 Slight No more than 20% of the cells areround, loosely attached, and without intracytoplasmic granules;occasional lysed cells are present 2 Mild Not more than 50% of the cellsare round and devoid of intracytoplasmic granules; no extensive celllysis and empty areas between cells 3 Moderate Not more than 70% of thecell layers contain rounded cells or are lysed 4 Severe Nearly completedestruction of the cell layers

6.83 Intramuscular Implantation Test—ISO—4 Week Implantation

6.8.3.1. Materials and Methods

To evaluate the potential of the test article to induce local toxiceffects, the Intramuscular Implantation Test—ISO—4 Week Implantation(“Intramuscular Implantation Test”) was used. Briefly, the test articlewas implanted in the paravertebral muscle tissue of New Zealand Whiterabbits for a period of 4 weeks. The test article was then evaluatedseparately using two control articles: positive control Surgicel(Johnson and Johnson, NJ) and negative control High Density Polyethylene(Negative Control Plastic).

Preparation of Test and Control Articles.

The test article measured approximately 1 mm to in width and 10 mm inlength. The two control articles were prepared. The positive control,Surgicel (C1), measured approximately 1 mm in width by 10 mm in lengthand was received sterile. Negative Control Plastic (C2), measuredapproximately 1 mm in width by 10 mm in length and was sterilized bydipping in 70% ethanol.

Pre-Dose Procedure.

Each animal was weighed prior to implantation. On the day of the test,the dorsal sides of the animals were clipped free of fur and loose hairwas removed by means of a vacuum. Each animal was appropriatelyanesthetized. Prior to implantation, the area was swabbed with asurgical preparation solution.

Dose Administration.

Four test article strips were surgically implanted into each of theparavertebral muscles of each rabbit, approximately 2.5 cm from themidline and parallel to the spinal column and approximately 2.5 cm fromeach other. The test article strips were implanted on one side of thespine. In a similar fashion, positive control article strips (Surgicel)were implanted in the contralateral muscle of each animal. Two negativecontrol strips (Negative Control Plastic) were implanted caudal (towardthe tail) to the test article and to C1 control implant sites on eitherside of the spine (total of four strips). A total of at least eight testarticle strips and eight of each control article strips are required forevaluation.

Post-Dose Procedures.

The animals were maintained for a period of 4 weeks. The animals wereobserved daily for this period to ensure proper healing of the implantsites and for clinical signs of toxicity. Observations included allclinical manifestations. At the end of the observation period, theanimals were weighed. Each animal was sacrificed by an injectablebarbiturate. Sufficient time was allowed to elapse for the tissue to becut without bleeding.

Gross Observations.

The paravertebral muscles in which the test or control articles wereimplanted were excised in toto from each animal. The muscle tissue wasremoved by carefully slicing around the implant sites with a scalpel andlifting out the tissue. The excised implant tissues were examinedgrossly, but without using excessive invasive procedures that might havedisrupted the integrity of this tissue for histopathological evaluation.The tissues were placed in properly labeled containers containing 10%neutral buffered formalin.

Histopathology.

Following fixation in formalin, each of the implant sites was excisedfrom the larger mass of tissue. The implant site, containing theimplanted material, was examined macroscopically. Each site was examinedfor signs of inflammation, encapsulation, hemorrhaging, necrosis, anddiscoloration using the following scale:

-   -   0=Normal    -   1=Mild    -   2=Moderate    -   3=Marked        After macroscopic observation, the implant material was left        in-situ and a slice of tissue containing the implant site was        processed. Histologic slides of hematoxylin and eosin stained        sections were prepared by Toxikon. The slides were evaluated and        graded by light microscopic examination.

Pathological Assessment of the Effects of the Implant.

The following categories of biological reaction were assessed bymicroscopic observation for each implant site:

-   -   1. Inflammatory Responses:        -   a. Polymorphonuclear leukocytes        -   b. Lymphocytes        -   c. Eosinophils        -   d. Plasma cells        -   e. Macrophages        -   f. Giant cells        -   g. Necrosis        -   h. Degeneration    -   2. Healing Responses:        -   a. Fibrosis        -   b. Fatty Infiltrate

Each category of response was graded using the following scale:

-   -   0=Normal    -   0.5=Very Slight    -   1=Mild    -   2=Moderate    -   3=Marked

The relative size of the involved area was scored by assessing the widthof the area from the implant/tissue interface to unaffected areas whichhave the characteristics of normal tissue and normal vascularity.Relative size of the involved area was scored using the following scale:

-   -   0=0 mm, No site    -   0.5=up to 0.5 mm, Very slight    -   1=0.6-1.0 mm, Mild    -   2=1.1-2.0 mm, Moderate    -   3=>2.0 mm, Marked

The Intramuscular Implantation Test was conducted based upon thefollowing

REFERENCES

-   1. ISO 10993-6, 1994, Biological Evaluation of Medical Devices—Part    6: Tests for Local Effects After Implantation.-   2. ISO 10993-12, 2002, Biological Evaluation of Medical Devices—Part    12: Sample Preparation and Reference Materials.-   3. ASTM F981-04, 2004, Standard Practice for Assessment of    Compatibility of Biomaterials for Surgical Implants with Respect to    Effect of Materials on Muscle and Bone.-   4. ASTM F763-04, 2004, Standard Practice for Short Term Screening of    Implant Materials.-   5. ISO/IEC 17025, 2005, General Requirements for the Competence of    Testing and Calibration Laboratories.

The results of the Intramuscular Implantation Test were evaluated basedupon the following criteria:

1. Calculated Rating: For each implanted site, a total score isdetermined. The average score of the test sites for each animal iscompared to the average score of the control sites for that animal. Theaverage difference between test and control sites for all animals iscalculated and the initial Bioreactivity Rating is assigned as follows:

-   -   0-1.5 No Reaction*    -   >1.5-3.5 Mild Reaction    -   >3.5-6.0 Moderate Reaction    -   >6.0 Marked Reaction    -   * A negative calculation is reported as zero (0).

2. Modification of the Rating: The pathology observer reviews thecalculated level of bioreactivity. Based on the observation of allfactors (e.g., relative size, pattern of response, inflammatory vs.resolution), the pathology observer may revise the Bioreactivity Rating.Justification for the modification to the rating is presented in thenarrative report (A descriptive narrative report regarding thebiocompatibility of the test material is provided by the pathologyobserver).

6.8.3.2. Results

The results indicated that the test article was non-reactive whenimplanted for 4 weeks (Bioreactivity Rating of 0.2) when compared topositive control Surgicel; and non-reactive (Bioreactivity Rating of0.0) when compared to negative control High Density Polyethylene(Negative Control Plastic).

Clinical Observation.

Table XI below shows results of the macroscopic evaluation of the testarticle and control implant sites indicated no significant signs ofinflammation, encapsulation, hemorrhage, necrosis, or discoloration atthe 4 week time period. Some test sites and the majority of the positivecontrol, Surgicel, were not seen macroscopically and serial sectionswere submitted for microscopic evaluation.

TABLE XI Macroscopic Observations 4 Week Implantation Tissue Size:Control Control Test C1 C2 T1 T2 T3 T4 Ave. C1-1 C1-2 C1-3 C1-4 Ave.C2-1 C2-2 C2-3 C2-4 Ave Animal No. 60959 Inflamation 0 NSF 0 NSF 0 NSFNSF NSF NSF N/A 0 0 0 0 0 Encapsulation 0 NSF 0 NSF 0 NSF NSF NSF NSFN/A 0 0 0 0 0 Hemorrhage 0 NSF 0 NSF 0 NSF NSF NSF NSF N/A 0 0 0 0 0Necrosis 0 NSF 0 NSF 0 NSF NSF NSF NSF N/A 0 0 0 0 0 Discoloration 0 NSF0 NSF 0 NSF NSF NSF NSF N/A 0 0 0 0 0 Total 0 N/A 0 N/A N/A N/A N/A N/A0 0 0 0 Animal No. 60961 Inflamation NSF NSF NSF NSF N/A NSF NSF NSF NSFN/A 0 0 NSF 0 0 Encapsulation NSF NSF NSF NSF N/A NSF NSF NSF NSF N/A 00 NSF 0 0 Hemorrhage NSF NSF NSF NSF N/A NSF NSF NSF NSF N/A 0 0 NSF 0 0Necrosis NSF NSF NSF NSF N/A NSF NSF NSF NSF N/A 0 0 NSF 0 0Discoloration NSF NSF NSF NSF N/A NSF NSF NSF NSF N/A 0 0 NSF 0 0 TotalN/A N/A N/A N/A N/A N/A N/A N/A 0 0 N/A 0 Animal No. 60968 InflamationNSF NSF NSF NSF N/A NSF NSF NSF NSF N/A 0 0 0 0 0 Encapsulation NSF NSFNSF NSF N/A NSF NSF NSF NSF N/A 0 0 0 0 0 Hemorrhage NSF NSF NSF NSF N/ANSF NSF NSF NSF N/A 0 0 0 0 0 Necrosis NSF NSF NSF NSF N/A NSF NSF NSFNSF N/A 0 0 0 0 0 Discoloration NSF NSF NSF NSF N/A NSF NSF NSF NSF N/A0 0 0 0 0 Total N/A N/A N/A N/A N/A N/A N/A N/A 0 0 0 0 T = test site(representative sections were submitted for microspoic assessment) C1 =Surgical (Due to the nature of the material, representative sectionswere submitted for microscopic assessment) C2 = Negative Control HighDensity Polyethylene (Negative Control Plastic) Grading Scale 0 = noreaction 1 = mild reaction 2 = moderate reaction 3 = marked reaction NSF= No Site Found N/A = Not Applicable

Implantation Site Observations (Microscopic).

Table XII below shows results of the microscopic evaluation of the testarticle implant sites indicated no significant signs of inflammation,fibrosis, hemorrhage, necrosis, or degeneration as compared to each ofthe control article sites. The Bioreactivity Rating for the 4 week timeperiod (average of three animals) was 0.2, (C1—Surgicel) and 0.0(C2—Negative Control Plastic) indicating no reaction as compared toeither of the control implant sites. The pathologist noted there was amoderate polymorphic and histiocytic (macrophages) infiltrate around thein situ test article that was not unexpected given the nature of thetest material.

TABLE XII Macroscopic Observations 4 Week Implantation Animal No.: 60959Categories Test Sites** Control Sites Reaction T1 T2 T3 C1-1 C1-2 C1-3C1-4 C2-1 C2-2 C2-3 C2-4 Foreign Debris 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 Rel. Size of Involved area 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.50.5 0.5 0.5 * Polymorphs 0.0 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 *Lymphocutes 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Eosinophils0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Plasma Cells 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Macrophages 0.5 0.5 0.5 0.5 0.5 0.50.5 0.5 0.5 0.5 0.5 * Giant Cells 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 * Degeneration 0.5 0.5 0.5 0.5 0.0 0.5 0.0 0.0 0.0 0.0 0.0 *Necrosis 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Fibrosis 0.5 0.50.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 * Fatty Infiltrate 0.0 0.0 0.5 0.00.5 0.0 0.5 0.5 0.5 0.0 0.5 Total 1.5 2.0 2.5 1.5 1.5 1.5 1.5 1.5 1.51.0 1.5 T = Test Site C1 = Surgical C2 = Negative Control High DensityPolyethylene (Negative Control Plastic) Animal Test Score (Average*) =2.0 Animal C1 Score (Average*) = 1.5 Animal C2 Score (Average*) = 1.4Animal Score (Average Test Score − Average C1 Score) = 0.5 Animal Score(Average Test Score − Average C2 Score) = 0.6 * Unused in calculation ofBioreactivity Rating **No site found in T4. Animal No.: 60961 CategoriesTest Sites** Control Sites** Reaction T1 T3 T4 C1-1 C1-3 C1-4 C2-1 C2-2C2-3 Foreign Debris 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Rel. Size ofInvolved area 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 * Polymorphs 0.0 0.00.5 0.5 0.0 0.5 0.5 0.5 0.5 * Lymphocutes 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 * Eosinophils 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Plasma Cells0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Macrophages 0.5 0.5 0.5 0.0 0.50.5 0.5 0.5 0.5 * Giant Cells 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 *Degeneration 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 * Necrosis 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 * Fibrosis 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 *Fatty Infiltrate 0.0 0.0 0.0 0.5 0.0 0.5 0.5 0.5 0.5 Total 1.5 2.0 2.02.5 1.5 2.5 2.5 2.5 2.5 T = Test Site C1 = Surgical C2 = NegativeControl High Density Polyethylene (Negative Control Plastic) Animal TestScore (Average*) = 1.8 Animal C1 Score (Average*) = 2.2 Animal C2 Score(Average*) = 2.5 Animal Score (Average Test Score − Average C1 Score) =−0.4 Animal Score (Average Test Score − Average C2 Score) = −0.7 *Unused in calculation of Bioreactivity Rating **No site found in T2,C1-2, and C2-4. Animal No.: 60968 Categories Test Sites Control Sites**Reaction T1 T2 T3 T4 C1-1 C1-2 C1-3 C2-1 C2-2 C2-3 C2-4 Foreign Debris0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Rel. Size of Involved area0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 * Polymorphs 0.0 0.5 0.0 0.50.0 0.0 0.0 0.5 0.5 0.0 0.5 * Lymphocutes 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 * Eosinophils 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00.0 * Plasma Cells 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 *Macrophages 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 * Giant Cells0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 * Degeneration 0.5 0.5 0.50.0 0.0 0.5 0.5 0.5 0.5 0.5 0.5 * Necrosis 0.0 0.0 0.0 0.5 0.0 0.0 0.00.0 0.0 0.0 0.0 * Fibrosis 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 *Fatty Infiltrate 0.5 0.5 0.5 0.5 0.5 0.0 0.5 0.5 0.5 0.5 0.5 Total 2.02.5 2.0 2.5 2.0 1.5 2.0 2.5 2.5 2.0 2.5 T = Test Site C1 = Surgical C2 =Negative Control High Density Polyethylene (Negative Control Plastic)Animal Test Score (Average*) = 2.3 Animal C1 Score (Average*) = 1.8Animal C2 Score (Average*) = 2.4 Animal Score (Average Test Score −Average C1 Score) = 0.5 Animal Score (Average Test Score − Average C2Score) = −0.1 * Unused in calculation of Bioreactivity Rating **No sitefound in C1-4. C1 C2 Animal Score 60959 = 0.5 0.6 Animal Score 60961 =−0.4 −0.7 Animal Score 60968 = 0.5 −0.1 Bioreactivity Rating = 0.2 = Noreaction Bioreactivity Rating = −0.1 = No reaction

6.8.4 Intracutaneous Injection Test—ISO 10993-10

USP 0.9% Sodium Chloride for Injections (NaCl) and Cottonseed Oil (CSO)extracts of the test article were evaluated for their potential toproduce irritation after intracutaneous injection in New Zealand Whiterabbits. The test article sites did not show a significantly greaterbiological reaction than the sites injected with the control article.Based on the criteria of the protocol, the test article is considered anegligible irritant and meets the requirements of the ISO 10993-10guidelines. Results are shown below in Table XIII.

TABLE XIII Intracutaneous Test Skin Reaction Scores Site Numbers Scoring(ER/ED) Animal # Vehicle Time T-1 T-2 T-3 T-4 T-5 C-1 C-2 C-3 C-4 C-5NaCl Extract 61917 NaCl  0 hours^(†) 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hours 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 61919 NaCl  0 hours^(†) 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/024 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hours 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 Total 0.0 0.0 CSO Extract 61917 CSCO  0 hours^(†) 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/048 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hours 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 61919 CSO  0 hours^(†) 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 24 hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48hours 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hours 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 Total 0.0 0.0 ^(†)= Immediately after injection,not used for the evaluation criteria. Overall Mean Score* for TestArticle = 0.0 Overall Mean Score* for Control Article = 0.0 Differencebetween Test Article and Control Article Overall Mean Score = 0.0 − 0.0= 0.0 ER = Erythema; ED = Edema; T = Test Sites; C = Control Sites*Overall Mean Score = Total erythema plus edema scores divided by 12 (2animals × 3 scoring periods × 2 scoring categories)

6.8.5 Kligman Maximization Test—ISO 10993-10

UPS 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO)extracts of the test article elicited no intradermal reaction in Hartleyguinea pigs at the challenge (0% sensitization), following an inductionphase. Therefore, as defined by the scoring system of Kligman, this is aGrade I reaction and the test article is classified as having weakallergenic potential. Based on the criteria of the protocol, a Grade Isensitization rate is not considered significant and the test articlemeets the requirements of the ISO 10993-10 guidelines. Results are shownbelow in Table XIV.

TABLE XIV Skin Examination Data Percent Scores Animals Allergenic GroupAnimal # Sex Day 25 Day 26 Day 27 Sensitized Potential Test Article 1Male 0 0 0 0% Weak (NaCl Extract) 2 Male 0 0 0 3 Male 0 0 0 4 Male 0 0 05 Male 0 0 0 6 Female 0 0 0 7 Female 0 0 0 8 Female 0 0 0 9 Female 0 0 010 Female 0 0 0 Test Article 11 Male 0 0 0 0% Weak (CSO Extract) 12 Male0 0 0 13 Male 0 0 0 14 Male 0 0 0 15 Male 0 0 0 16 Female 0 0 0 17Female 0 0 0 18 Female 0 0 0 19 Female 0 0 0 20 Female 0 0 0 Negative 21Male 0 0 0 0% Weak Control (NaCl) 22 Male 0 0 0 23 Female 0 0 0 24Female 0 0 0 25 Female 0 0 0 Negative 26 Mate 0 0 0 0% Weak Control(CSO) 27 Male 0 0 0 28 Female 0 0 0 29 Female 0 0 0 30 Female 0 0 0Positive Control 31 Male 2 1 0 100% Extreme (DNCB) 32 Male 2 2 1 33Female 3 2 1 34 Female 3 2 1 35 Female 3 3 2 Sensitization Rate (%)Grade Class 0-8 I Weak  9-28 II Mild 29-64 III Moderate 65-80 IV Strong 81-100 V Extreme The test results are interpreted based upon thepercentage sensitization observed.

7. INCORPORATION BY REFERENCE

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for treating (i) a bacterial infectionin a subject, or (ii) a disease associated with a bacterial infection ora bacterial imbalance in a subject, comprising: topically administeringa composition comprising sNAG nanofibers to a subject in need thereof,wherein (a) the sNAG nanofibers were produced by irradiation ofpoly-N-acetylglucosamine or a derivative thereof, and more than 50% ofthe sNAG nanofibers are no greater than 15 μm in length, or (b) the sNAGnanofibers were produced by irradiation of poly-N-acetylglucosamine or aderivative thereof with a dose of irradiation that reduces the averagelength of the sNAG nanofibers to less than 15 μm in length, and whereinthe sNAG nanofibers do not have an effect on bacterial growth orsurvival of Staphylococcus aureus bacterial cultures in vitro.
 2. Themethod of claim 1, wherein the bacterial infection is a skin infection,a gastrointestinal infection, a respiratory infection, a urinary tractinfection, or a reproductive tract infection.
 3. The method of claim 1,which is the method for treating a bacterial infection in a subject,wherein the subject is a subject diagnosed with the bacterial infection,or displaying one or more symptoms of the bacterial infection.
 4. Themethod of claim 3, wherein the bacterial infection is (i) not at thesite of a wound, (ii) not associated with or caused by a wound, or (iii)not at the site of a wound and not associated with or caused by a wound.5. The method of claim 3, wherein the bacterial infection is anosocomial infection.
 6. The method of claim 3, wherein the sNAGnanofibers are in an amount effective to achieve one or more of thefollowing: (i) reduce the severity of the bacterial infection or one ormore symptoms of the bacterial infection, (ii) reduce the duration ofthe bacterial infection or one or more symptoms of the bacterialinfection, and (iii) eradicate the bacterial infection or one or moresymptoms of the bacterial infection.
 7. The method of claim 1, which isthe method for treating the disease, wherein the disease is associatedwith a bacterial infection, and where the subject is a subject diagnosedwith the disease or displaying one or more symptoms of the disease. 8.The method of claim 7, wherein the disease is a skin disease or agastrointestinal disease.
 9. The method of claim 7, wherein thebacterial infection is (i) not at the site of a wound, (ii) notassociated with or caused by a wound, or (iii) not at the site of awound and not associated with or caused by a wound.
 10. The method ofclaim 7, wherein the sNAG nanofibers are in an amount effective toachieve one or more of the following: (i) reduce the severity of thedisease or one or more symptoms of the disease, (ii) reduce the durationof the disease or one or more symptoms of the disease, and (ii) preventthe progression of the disease or one or more symptoms of the disease.11. The method of claim 1, which is the method for treating the disease,wherein the disease is associated with a bacterial imbalance, and wherethe subject is a subject diagnosed with the disease or displaying one ormore symptoms of the disease.
 12. The method of claim 1, wherein thesNAG nanofibers are non-reactive when tested in an elution test, anintramuscular implantation test, an intracutaneous test, and/or asystemic test.
 13. The method of claim 1, wherein the composition doesnot comprise an additional anti-bacterial agent.
 14. The method of claim13, wherein the composition is not administered in conjunction with ananti-bacterial agent.
 15. The method of claim 14, wherein thecomposition is not administered with an immunomodulator.
 16. A methodfor treating a bacterially infected wound in a subject, comprising:topically administering a composition comprising sNAG nanofibers to thewound site in a subject diagnosed with the bacterial infection ordisplaying one or more symptoms of the bacterial infection, wherein (a)the sNAG nanofibers were produced by irradiation ofpoly-N-acetylglucosamine or a derivative thereof, and more than 50% ofthe sNAG nanofibers are no greater than 15 μm in length, or (b) the sNAGnanofibers were produced by irradiation of poly-N-acetylglucosamine or aderivative thereof with a dose of irradiation that reduces the averagelength of the sNAG nanofibers to less than 15 μm in length, wherein thesNAG nanofibers do not have an effect on bacterial growth or survival ofStaphylococcus aureus bacterial cultures in vitro, wherein thecomposition does not comprise an additional anti-bacterial agent, andwherein the composition is not administered in conjunction with ananti-bacterial agent.
 17. The method of claim 16, wherein the wound isan open wound.
 18. The method of claim 17, wherein the open wound is agunshot wound, a puncture wound, a laceration wound, a cut, an abrasion,a penetration wound or a surgical wound.
 19. The method of claim 17,wherein the open wound is a puncture wound, wherein the puncture woundis caused by a hemodialysis procedure or a catheterization procedure,and wherein the subject has been diagnosed with a hemodialysis-relatedor catheterization-related infection.
 20. The method of claim 16,wherein the sNAG nanofibers are in an amount effective to achieve one ormore of the following: (i) reduce the severity of the bacterialinfection or one or more symptoms of the bacterial infection, (ii)reduce the duration of the bacterial infection or one or more symptomsof the bacterial infection, and (iii) eradicate the bacterial infectionor one or more symptoms of the bacterial infection.
 21. The method ofclaim 16, wherein the sNAG nanofibers are non-reactive when tested in anelution test, an intramuscular implantation test, an intracutaneoustest, and/or a systemic test.
 22. The method of claim 16, wherein thecomposition is not administered with an immunomodulator.
 23. The methodof claim 1 or 16, wherein the composition does not comprise anadditional therapy which is encapsulated, immobilized or formulated inthe sNAG nanofibers.
 24. The method of claim 1 or 16, wherein thecomposition does not comprise an additional active ingredient.
 25. Themethod of claim 24, wherein the composition is not administered inconjunction with an additional active ingredient.
 26. The method ofclaim 1 or 16, wherein the bacterial infection is an infection with abacteria of at least one of the following species: Bacillus anthracis,Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucellacanis, Brucella melitensis, Brucella suis, Campylobacter jejuni,Chlamydia pneumonia, Chlamydia trachomatis, Clamidophila psittaci,Clostridium botulinum, Clostridium difficule, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Francisella tularensis,Haemophilus influenae, Helicobacter pylori, Legionella pneumphila,Leptospira pneumophila, Leptospira interrogans, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa,Proteus mirabilis, Rickettsia rickettsii, Salmonella typhi, Salmonellatyphimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum,Vibria cholerae, and Yersinia pestis.
 27. The method of claim 1 or 16,wherein the bacteria is resistant to a standard antibiotic therapy. 28.The method of claim 1 or 16, wherein the bacterial infection is aMethycillin-resistant Staphylococcus aureus infection, a Pseudomonasinfection, or a C. dificule infection.
 29. The method of claim 1 or 16,wherein the subject is a human.
 30. The method of claim 1 or 16, whereinthe sNAG nanofibers are formulated as a cream, a gel, an ointment, amembrane, a powder, a spray, or a suppository.
 31. The method of claim 1or 16, wherein more than 50% of the sNAG nanofibers are less than about10 μm in length as determined by scanning electron microscopic (SEM)analysis.
 32. The method of claim 1 or 16, wherein more than 50% of thesNAG nanofibers are between about 1 to 8 μm in length as determined byscanning electron microscopic (SEM) analysis.
 33. The method of claim 1or 16, wherein (i) the poly-N-acetylglucosamine or a derivative thereofwas irradiated in the form of dry fibers, a dry fiber membrane or a drylyophilized material at 500-2,000 kgy, or (ii) thepoly-β-N-acetylglucosamine or a derivative thereof was irradiated in theform of a suspension, a slurry or a wet cake at 100-500 kgy.
 34. Themethod of claim 1 or 16, wherein (i) the poly-N-acetylglucosamine or aderivative thereof was irradiated by gamma irradiation in the form ofdry fibers, a dry fiber membrane or a dry lyophilized material at750-1,250 kgy, or (ii) the poly-β-N-acetylglucosamine or a derivativethereof was irradiated by gamma irradiation in the form of a suspension,a slurry or a wet cake at 150-250 kgy.
 35. The method of claim 1 or 16,wherein the sNAG nanofibers were produced from a microalgalpoly-N-acetylglucosamine.
 36. The method of claim 1 or 16, wherein thesNAG nanofibers comprise N-acetylglucosamine monosaccharides and/orglucosamine monosaccharides, and wherein more than 70% of themonosaccharides of the sNAG nanofibers are N-acetylglucosaminemonosaccharides.
 37. The method of claim 1 or 16, wherein the sNAGnanofibers comprise N-acetylglucosamine monosaccharides and/orglucosamine monosaccharides, and wherein more than 95% of themonosaccharides of the sNAG nanofibers are N-acetylglucosaminemonosaccharides.
 38. The method of claim 1 or 16, wherein the sNAGnanofibers do not elicit a detectable foreign body reaction.
 39. Themethod of claim 1 or 16, wherein the composition comprises 0.2 to 20mg/cm² of the sNAG nanofibers per dose or application of thecomposition.
 40. The method of claim 1 or 16, wherein the sNAGnanofibers increase the metabolic rate of serum-starved human umbilicalcord vein endothelial cells in a MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.41. The method of claim 40, wherein the sNAG nanofibers do not rescueapoptosis of serum-starved human umbilical cord vein endothelial cellsin a trypan blue exclusion test.
 42. The method of claim 1 or 16,wherein the infrared spectrum (“IR”) of the sNAG nanofibers is similarto, about the same as, or equivalent to that of non-irradiatedmicroalgal poly-N-acetylglucosamine.
 43. The method of claim 1 or 16,wherein the infrared spectrum (“IR”) of the sNAG nanofibers isequivalent to that of non-irradiated microalgalpoly-N-acetylglucosamine.
 44. The method of claim 1 or 16, wherein thesNAG nanofibers have the chemical and physical structure of the fibersas determined by infrared (IR) spectrum, elemental assay and scanningelectron microscopic (SEM) analyses.
 45. A method for treating abacterial infection in a human subject, comprising: topicallyadministering a composition comprising an effective amount of sNAGnanofibers to a human subject diagnosed with the bacterial infection ordisplaying one or more symptoms of the bacterial infection, wherein (a)more than 50% of the sNAG nanofibers are less than 15 μm in length, or(b) the sNAG nanofibers were produced by irradiation ofpoly-N-acetylglucosamine or a derivative thereof with a dose ofirradiation that reduces the average length of the sNAG nanofibers toless than 15 μm in length, wherein the sNAG nanofibers have themicrostructure of the fibers, wherein the sNAG nanofibers compriseN-acetylglucosamine monosaccharides and/or glucosamine monosaccharides,and wherein more than 70% of the monosaccharides of the sNAG nanofibersare N-acetylglucosamine monosaccharides, wherein the sNAG nanofibers donot have an effect on bacterial growth or survival of Staphylococcusaureus bacterial cultures in vitro, wherein the bacterial infection is askin infection, a gastrointestinal infection, a respiratory infection, aurinary tract infection, or a reproductive tract infection, and whereinthe composition does not comprise an additional anti-bacterial agent orimmunomodulator, and is not administered in conjunction with anadditional anti-bacterial agent or immunomodulator.
 46. The method ofclaim 45, wherein the sNAG nanofibers were produced by gamma irradiationof poly-N-acetylglucosamine or a derivative thereof, and wherein (i) thepoly-N-acetylglucosamine or a derivative thereof was irradiated in theform of dry fibers, a dry fiber membrane or a dry lyophilized materialat 500-2,000 kgy, or (ii) the poly-β-N-acetylglucosamine or a derivativethereof was irradiated in the form of a suspension, a slurry or a wetcake at 100-500 kgy.
 47. The method of claim 45, wherein more than 50%of the sNAG nanofibers are between about 1 and 15 μm in length asdetermined by scanning electron microscopic (SEM) analysis.
 48. Themethod of claim 46 or 47, wherein the bacterial infection is aPseudomonas infection.
 49. The method of claim 46 or 47, wherein thebacterial infection is a Staphylococcus infection.
 50. The method ofclaim 45, wherein the sNAG nanofibers comprise N-acetylglucosaminemonosaccharides and/or glucosamine monosaccharides, and wherein morethan 95% of the monosaccharides of the sNAG nanofibers areN-acetylglucosamine monosaccharides.
 51. The method of claim 45, whereinthe sNAG nanofibers were produced from a microalgalpoly-N-acetylglucosamine.
 52. The method of claim 1 or 16, wherein theirradiation is gamma irradiation.
 53. The method of claim 1 or 16,wherein the sNAG nanofibers were produced by irradiation ofpoly-N-acetylglucosamine or a derivative thereof, and more than 50% ofthe sNAG nanofibers are no greater than 15 μm in length as determined byscanning electron microscopic (SEM) analysis.
 54. The method of claim 1or 16, wherein the sNAG nanofibers were produced by irradiation ofpoly-N-acetylglucosamine or a derivative thereof with a dose ofirradiation that reduces the average length of the sNAG nanofibers toless than 15 μm in length.
 55. A method for treating (i) a bacterialinfection in a subject, or (ii) a disease associated with a bacterialinfection or a bacterial imbalance in a subject, comprising: topicallyadministering a composition comprising an effective amount of sNAGnanofibers to a subject in need thereof, wherein the sNAG nanofiberswere produced by irradiation of poly-N-acetylglucosamine or a derivativethereof with a dose of irradiation that reduces the average length ofthe sNAG nanofibers to less than 15 μm in length, wherein (i) the sNAGnanofibers have the microstructure of the fibers; (ii) the sNAGnanofibers have the chemical and physical structure of the fibers asdetermined by infrared spectrum, elemental assay and scanning electronmicroscopic (SEM) analyses; and/or (iii) the infrared spectrum of thesNAG nanofibers is equivalent to that of non-irradiated microalgalpoly-N-acetylglucosamine, and wherein the sNAG nanofibers do not have aneffect on bacterial growth or survival of Staphylococcus aureusbacterial cultures in vitro.
 56. The method of claim 55, wherein thecomposition does not comprise an additional anti-bacterial agent, andwherein the composition is not administered in conjunction with ananti-bacterial agent or an immunomodulator.
 57. The method of claim 55,wherein the bacterial infection is a Pseudomonas infection, aStaphylococcus infection, or a C. difficule infection.
 58. The method ofclaim 57, wherein the bacterial infection is a Pseudomonas aeruginosainfection or a Staphylococcus aureus infection.
 59. The method of claim55, wherein the sNAG nanofibers comprise N-acetylglucosaminemonosaccharides and/or glucosamine monosaccharides, and wherein morethan 70% of the monosaccharides of the sNAG nanofibers areN-acetylglucosamine monosaccharides.
 60. The method of claim 55, whereinthe sNAG nanofibers were produced from a microalgalpoly-N-acetylglucosamine.
 61. The method of claim 55, wherein thesubject is a human.
 62. The method of claim 55, wherein the irradiationis gamma irradiation.
 63. The method of claim 1, 16, 45 or 55, whereinthe poly-N-acetylglucosamine or a derivative thereof ispoly-N-acetylglucosamine that comprises at least 70% ofN-acetylglucosamine monosaccharides.